Mammalian neuralized family transcriptional regulators and uses

ABSTRACT

The disclosure relates to isolated polynucleotides and purified polypeptides of the Neu family of proteins, which have been shown to demonstrate transcriptional regulatory activity. For example, the purified polynucleotide can encode a Neu polypeptide, wherein the Neu polypeptide comprises at least one neuralized homology repeat domain and a C3HC4 RING-zinc finger domain is disclosed. A purified Neu polypeptide, wherein the Neu polypeptide comprises at least one neuralized homology repeat domain and a C3HC4 RING-zinc finger domain is disclosed. Antibodies capable of specifically binding to the disclosed Neu polypeptides are disclosed. Vectors expressing the disclosed Neu protein coding regions and host cells containing the vectors are disclosed. Methods of making the Neu proteins disclosed are also provided, as are method of identifying binding partners that interact with a Neu protein family member.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The described invention relates to the family of mammalian Neuralized(neu) genes, proteins encoded by those genes (Neu), expression patternsof the gene family, their function as a transcriptional regulator, andthe proteins with which the Neu family of proteins interacts.Additionally, therapeutic and diagnostic uses for the Neu family ofproteins and agents that bind thereto are also provided.

2. Description of the Related Art

Development and functioning of the nervous system requires orchestratedaction of thousands of transcriptional regulators. Balance betweentranscriptional activators and repressors determines the spectrum ofexpressed genes. The molecular basis of the initial stages ofneurogenesis as well as several aspects of neuronal differentiation havebeen extensively studied. As a result, a variety of transcriptionalactivators and repressors have been discovered and characterized. Ourunderstanding of these systems and their component interactions,however, is far from complete. Little is known about the molecularmechanisms that support neuronal circuits in developing and maturenervous systems or the molecular mechanisms that coordinate maintenanceof the differentiated state.

The process of lateral inhibition prevents neighboring cells fromdeveloping into the same type of differentiated cells in flies and invertebrates. Tanabe & Jessell, Science, 274:1115-23 (1996). InDrosophila, a group of mutations has been described that shows severedefects in the process of lateral inhibition in the developing nervoussystem. These neurogenic mutations result in hyperplasia of the neuraltissue at the expense of epidermal structures. Campos-Ortega & Jan, AnnuRev Neurosci, 14:399-420 (1991). The temporal and spatial expressionpatterns of the neu are compatible with its function as a neurogenicgene in Drosophila. The Neu protein is expressed throughout the ectodermat the time when cell fate is determined and its expression proceeds inneuroblasts. Boulianne, et al., EMBO J, 10:2975-2983 (1991). neuexpression has been detected in actively proliferating neuroblasts inseveral regions of the CNS and PNS. Expression of neu in imaginal discsuggests that it is also involved in later stages of development. Theneu gene encodes a RING finger (C3HC4) type zinc finger protein. Themolecular function of the Drosophila Neu protein is unknown.Interestingly, it was discovered that EST data bases contain a homologueof Neu suggesting that a family of Neu-like proteins is present inDrosophila.

Current studies of the brain development in Drosophila and vertebrates,indicate that many basic molecular and genetic mechanisms involved inneurogenesis are highly conserved. During development of the nervoussystem, neural cell specification is acquired through the series ofprogressive restrictive steps. In Drosophila, neural precursors arefirst specified by proneural genes including basic helix-loop-helix(bHLH) transcription factors of atonal and achaete-scute complex.Simpson, Neuron, 15:739-742 (1995). The process of lateral inhibition,which further restricts the developmental potential of neuroectodermalcells is regulated by neurogenic genes such as Notch, mastermind, bigbrain, Delta, Enhancer of split, and neuralized. Analysis of thefunction of these neurogenic loci in the Drosophila embryo has revealedthat mutations in any of these genes result in hyperplasia of neuraltissue at the expense of epidermal structures and also cause defects intissues derived from mesoderm and endoderm. Campos-Ortega and Jan, Annu.Rev Neurosci, 14:399-420 (1991); Harentstein et al., Development,116:1203-1220 (1992). Vertebrate homologues of Notch, Delta and theproneural/neurogenic genes of atonal, achaete-scute, hairy, and Enhancerof Split complex have been identified and recent work, mostly in Xenopusand mouse, suggests that their role in neurogenesis is conserved. Lewis,Curr Opin Neurobiol, 6:3-10 (1996); Kageyama et al., Int J Biochem CellBiol, 29:1389-1399 (1997); and Beatus, Lendahl, J Neurosci Res54:125-136 (1998). For example, postnatal Notch signaling affects theelaboration of different body systems and regulates plasticity ofcortical postmitotic neurons. Artavanis-Tsakonas et al., Science,284:770-776 (1999); Redmond et al., Nat Neurosci, 3:30-40 (2000); andSestan et al., Science, 286:741-746 (1999).

The last few years have brought the identification and characterizationof many new key regulators of vertebrate neurogenesis. Recently, a humanhomologue of Drosophila Neu gene was isolated and its expression in theadult nervous system and in tumors of neuroectodermal origin, such asastrocytomas, was characterized. Nakamura, et al., Oncogene16(8):1009-1019 (1998). Nakamura and others (1998) hypothesized thath-neu1 plays a role in determination of cell fate in the central nervoussystem and may act as a tumor suppressor which inactivation could beassociated with malignant progression of astrocytic tumors. A homologysearch in human, rat, and mouse EST databases revealed three newmammalian Neu homologs, suggesting that a family of Neu-like proteinsexists in mammals.

SUMMARY OF THE INVENTION

The disclosure relates to isolated polynucleotides and purifiedpolypeptides of the Neu family of proteins, which have been shown todemonstrate transcriptional regulatory activity. For example, thepurified polynucleotide can encode a Neu polypeptide, wherein the Neupolypeptide comprises at least one neuralized homology repeat domain anda C3HC4 RING-zinc finger domain is disclosed. A purified Neupolypeptide, wherein the Neu polypeptide comprises at least oneneuralized homology repeat domain and a C3HC4 RING-zinc finger domain isdisclosed. Antibodies capable of specifically binding to the disclosedNeu polypeptides are disclosed. Vectors expressing the disclosed Neuprotein coding regions and host cells containing the vectors aredisclosed. Methods of making the Neu proteins disclosed are alsoprovided, as are methods of identifying binding partners that interactwith a Neu protein family member.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the primary structure of neu1 protein isoforms in mouse andrat.

FIG. 2 is an analysis of neu1 mRNA expression by RNase protection assayin mouse and rat.

FIG. 3 is an in situ hybridization analysis of neu1 mRNA expression inthe developing and adult mouse brain.

FIG. 4 is an in situ hybridization analysis of neu1 mRNA expression inadult rat brain.

FIG. 5 shows expression of neu1 mRNA in an adult rat nervous system.

FIG. 6 shows cellular localization of neu1 mRNA in an adult rat nervoussystem.

FIG. 7 shows the combined analyses of in situ hybridization of neu1 mRNAand immunohistochemistry of the neuronal marker neuN.

FIG. 8 shows neu1 mRNA expression in the hippocampus of adult rat brainafter kainic acid treatment.

FIG. 9 shows transcriptional analysis from various promoters intransient expression assays.

FIG. 10 shows that neuralized homology repeat domains of neu1 mediatethe transcriptional repression when fused to the DNA binding domain ofGal4.

FIG. 11 shows the subcellular localization of neu-FLAG and neu-EGFPfusion proteins in Neuro2A cells, using FLAG immunofluorescence usinganti-FLAG antibody (A, E, F, G) and direct fluorescence of EGFP fusionproteins (B, C, D, H).

FIG. 12 shows alignment of neuralized homology repeat domains of humanNeu1, Neu2, and Neu3 proteins.

FIG. 13 shows the analysis of neu2 mRNA expression by RNase protectionassay in mouse and rat.

FIG. 14 shows the analysis of neu3 mRNA expression by RNase protectionin mouse.

FIG. 15 is an analysis of the expression of rat Neu1 interactors byRNase protection assay.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention described herein relates to the identification of membersof the Neuralized (neu) family of genes and their respective proteins,and aspects of their structure and function as transcriptionalregulators. The invention relates to the characteristics of mammalianneu gene family's conserved structural motifs, related function astranscriptional regulators, and functional features of the familyrelating to expression patterns and potential interactions withinteraction-partners, providing tools with which to explore theNeu-related regulatory cascades that operate in neurons and other celltypes.

Structure

The invention described herein relates to the neuralized (Neu) family oftranscriptional regulators that contain a C-terminal C3HC4 RING zincfinger and at least one neuralized homology repeat domain. The term“neuralized” (or “Neu”), as used herein, includes all members of the Neufamily such as Neu1, Neu2, Neu3, Neu4 coding sequences and proteins.These proteins belong to a family of proteins that share C-terminalC3HC4 RING zinc finger and at least one neuralized homology repeatdomain. Neuralized homology repeat domains represent a novel class oftranscription repression domains that regulate transcription of a largenumber of genes.

The C3HC4 RING-zinc finger motif is a cystine-rich amino acid sequencemotif found in the sequence of the human RING gene. Freemont, et al.,Biochem J, 278:1-23 (1991); Freemont, Ann NY Acad Sci, 684:174-192(1993). The motif can be described asC—X₂—C—X₍₉₋₂₇₎—C—X₍₁₋₃₎—H—X₂—C—X₂—C—X₍₄₋₄₈₎—C—X₂—C, where C is acysteine, H is a histamine, and X can be any amino acid. This familyincludes genes that are involved in the regulation of development,differentiation, apoptosis, oncogenesis, and membrane trafficking.

Several isolated and characterized RING-zinc finger proteins haveproposed roles in gene regulation. Although the precise function of theRING-zinc finger domain is unknown, indirect data demonstrates that theRING-zinc finger domain could function as a DNA-binding orprotein-protein interaction domain. These two functions are related.Several transcriptional repressors which act as dimers have beencharacterized. Their structures comprise both dimerization andDNA-binding surfaces. For example, well known transcriptional regulatorsthat contain RING-zinc finger domains are BRCA1 (Miki et al., Science,266(5182):66-71 (1994)) and MEL18 (Kanno et al., EMBO J.15;14(22):5672-8 (1995)), a polycomb group-related transcriptionalregulator.

Function and Interactions

Neu Function as a Transcriptional Regulator

Mammalian Neu1 acts as a powerful transcriptional repressor in transientexpression assays and silences both, TATA-containing and TATA-lesspromoters, including the promoters of NGF, BDNF, NF-L, and GAP-43. NHRsfunction as transcription repression domains, suggesting thatNHR-containing proteins represent a novel class of transcriptionalrepressors. It is likely that mammalian Neu1 mediates transcriptionalrepression through protein-protein interactions. Like several knownrepressors, mammalian Neu1 could function through interaction withco-repressors such as dCtBP or mammalian homologues of Groucho, andgeneral repressor complexes, such as NC2, Mot1, or Not to interfere withthe function of Pol II complex. Maldonado et al., Cell, 99:455-458(1999) and Mannervik et al., Science, 284:606-609 (1999). Alternatively,repression could be achieved through chromatin remodeling by recruitingthe histone deacetylase complexes (HDACs). Glass and Rosenfeld, EndocrRev, 21:447 (2000); Knoepfler and Eisenman, Cell, 99:447-450 (1999); andTorchia et al., Curr Opin Cell Biol, 10:373-383 (1998). Current datasuggest that the mechanism of Neu1 repression does not include a HDACcomplex in neuroblastoma Neuro2A cells, as the HDAC inhibitortrichostatinA did not relieve m-Neu1-mediated repression in these cells.

Neu is a Shuttling Protein with Dominant Cytoplasmic Localization as aResult of a Nuclear Import Combined with an Efficient Export

A putative nuclear localization signal has been identified in theN-terminus of d-Neu, however, the NLS sequence identified in d-Neu, isnot conserved in mouse, rat and human Neu proteins. Boulianne et al.,EMBO J 10:2975-2983 (1991) and Price et al., EMBO J, 12:2411-2418(1993). The weak NLS sequences (HKAVKR (SEQ ID NO: 43), RLKITKK (SEQ IDNO: 44)), that were identified in mammalian Neu1 proteins, have beensuggested to regulate nuclear import of a fraction of the synthesizedprotein. Boulikas, J Cell Biochem, 60:61-82 (1996). Indeed, m-Neu1resides both in the cytoplasm and in the nucleus, revealing that it isthe subject of regulated nuclear import. Recent studies have shown thatimportin-α family members are involved in the formation of the NLSreceptor complexes that govern the protein transport to the nucleus.Ullman et al., Cell, 90:967-970 (1997); and Izaurralde and Adam, RNA,4:351-364 (1998). Interestingly, importin-α3 was identified here as oneof the m-Neu interacting proteins by yeast two-hybrid screening.Furthermore, the results discussed herein also demonstrate that theCRM1/exportin1-related export pathway controls the nucleocytoplasmicshuttling of Neu1, since the nuclear export of a tagged-m-Neu1 fusionprotein is blocked by LMB. LMB is a microbial metabolite thatinactivates the nuclear export by interfering with the binding ofCRM1/exportin1 to the nuclear export signals. Fornerod et al., Cell,90:1051-1060 (1997); Kudo et al., Exp Cell Res, 242:540-547 (1998); Kudoet al., Proc Natl Acad Sci USA, 96:9112-9117 (1999); Fukuda et al.,Nature, 390:308-311 (1997); and Nishi et al., J Biol Chem, 269:6320-6324(1994). These data reveal that mammalian Neu1 function is additionallyregulated by nucleocytoplasmic shuttling.

Neu Function as a Calcium-Signal Transducer

Transcriptional activity of Neu is controlled by calcium (Ca²⁺)signaling that regulates Neu translocation into the nucleus where Neuacts as a transcriptional repressor. It has been long known thatintracellular calcium controls a variety of brain- and muscle cellfunctions. Since Ca²⁺ levels regulate nucleocytoplasmic shuttling oftranscription factors (Crabtree, Cell, 96:611-614 (1999)), Neu proteinscould function as mediators of calcium signaling to the nucleus toregulate gene expression. There is evidence that in Drosophila and alsoin vertebrates, ligand activated Notch is subjected to proteolyticcleavage and transported to the nucleus where it acts as atranscriptional regulator (Jarriault et al 1995; Ohtsuka et al., 1999).Interestingly, recently it was shown that d-neu protein is associatedprimarily with the plasma membrane (Yeh et al., 2000). This, however,does not exclude the possibility that Neu1, like Notch, may be localizedalso within the nucleus. Based on current knowledge it is hypothesizedthat Neu1 functions as a mediator of an extracellular signal from theplasma membrane to the nucleus to regulate gene expression. According toone possible scenario, Neu1 is posttranslationally modified in a signal(Ca²⁺)-dependent fashion and subsequently translocated into the nucleus,where it functions as a transcriptional regulator.

Neurogenic Function of Neu.

Drosophila neuralized (d-neu) and h-neu1 genes encode homologous (˜40%)proteins with a C-terminal C3HC4 RING zinc finger domain (RZD) and oneor two neuralized homology repeat (NHR) domains. Boulianne et al., EMBOJ, 10:2975-2983 (1991) and Price et al., EMBO J, 12:2411-2418 (1993).d-neu is expressed in the ectoderm at the time when cell fate isdetermined, implying the role of Neu in neurogenesis. Boulianne et al.,EMBO J, 10:2975-2983 (1991).

In Drosophila, d-neu expression has been detected earliest in theectoderm continuing later in neuroblasts. Boulianne et al., EMBO J.10:2975-2983 (1991). Since developing mouse CNS can be divided intoregions that express either high or low levels of neu1, and as neu1 isnot expressed in proliferating regions of the nervous system, itsuggests that other neu-related genes function in a fashioncomplementary to neu1. Indeed, based on the recent findings we couldargue that neu1 defines a new gene and protein family consisting of atleast two Drosophila and four mammalian genes.neu2, second member ofmammalian neu family of genes, is expressed at high levels in theembryonic brain, whereas the expression levels decrease during postnataldevelopment. Recent studies of the brain development in Drosophila andvertebrates, indicate that many basic molecular and genetic mechanismsinvolved in neurogenesis are highly conserved. Lewis, J. Curr OpinNeurobiol, 6:3-10 (1996); Kageyama et al., Int J Biochem Cell Biol,29:1389-1399 (1997); and Beatus & Lendahl, J Neurosci Res, 54:125-136(1998). For example, postnatal Notch signaling affects the elaborationof different body systems and regulates plasticity of corticalpostmitotic neurons. Artavanis-Tsakonas et al., Science, 284:770-776(1999); Redmond et al., Nat Neurosci, 3:30-40 (2000); and Sestan et al.,Science, 741-746 (1999).

Earlier genetic studies in Drosophila suggested that delta, mastermind,big brain, and neuralized refine a signal upstream of notch and thatmastermind functions upstream of all the other neurogenic genes.Campos-Ortega and Jan, Annu Rev Neurosci, 14:399-420 (1991); and Lieber,Genes Dev, 7:1949-1965 (1993). Therefore, if the function of themammalian Neu family of proteins is conserved in conjunction with otherneurogenic factors, mammalian neu genes refine the signal upstream ofmammalian homologues of notch and downstream of mastermind in tissueswhere neu genes are expressed, particularly in developing nervoussystem. Given that mammalian neu mRNAs encode different proteinisoforms, complex regulatory circuits implicating various Neu familymembers are expected in different tissues. The transcription repressionactivities of mammalian Neu could well-explain its function as aneurogenic gene.

Neu Role in Cell Signalling and Synaptogenesis

Suppression of gene expression plays an important role in themaintenance and stability of a mature nervous system. It is essential tosuppress neurite growth and extensive formation of new axons anddendrites in the adult functional nervous system and to maintain neuronsin their differentiated state. Transcriptional repressors are involvedand play a crucial role in the silencing of the neurite growth program.

Mammalian neu1 shows most prominent expression in the postnatal centralnervous system, revealing its function in postnatal development. neu1mRNA expression levels increase significantly during the early postnataldevelopment when active synaptogenesis takes place, to reach the peaklevels in the adult animal. In the adult mammalian CNS, the highestexpression levels of neu1 assign to the neurons of hippocampus, cerebralcortex, striatum, and amygdala. Whereas several brain regions such asthalamus/hypothalamus, midbrain, medulla, and also the spinal cordexhibit low expression. This indicates to the independent regulation ofneu1 expression in various brain regions by specific signalingmechanisms as well as to the requirement of Neu function for differentcell-cell signaling systems.

neu1 mRNA expression studies in adult rat brain revealed that in severalbrain regions, particularly in the granular cells of dentate gyrus ofhippocampus, neu1 mRNA is localized in the dendrites, suggesting thatsynthesis of Neu1 protein also occurs in dendrites. Several dataindicate that proteins locally translated from dendritic mRNAs atactivated synapses provide basis for activity-dependent regulation ofsynaptic modulation (reviewed in Steward et al., Neuron, 21:741-51(1997); Kuhl and Skehel, Curr Opin Neurobiol, 8(5):600-6 (1998);Schuman, Neuron, 23:645-8 (1999); Tiedge et al., Science, 283:186-7(1999); Kiebler and DesGroseillers, Neuron, 25:19-28 (2000)).Accordingly, Neu could be involved in the regulation of neuritogenesisand/or synaptogenesis, affecting the generation of the precise patternof neuronal connectivity. Recent molecular perturbation experimentssuggested that Notch1 signalling in cortical neurons promotes dendriticbranching and inhibits neurite growth (Redmond et al., 2000; Sestan etal., 1999). Suggesting that the function of neu1 to refine a signalupstream of Notch in Drosophila is evolutionary conserved in mammaliannervous system, it is possible, that Neu and Notch pathways act in aninterrelated manner to confer developmental plasticity to adult neurons.The possible similarities in the molecular mechanisms of function of twogenes, Notch and neu1, that were first discovered as neurogenic, areappealingly apparent.

Neu Role Related to Repair and Regeneration After Injury to the CNS

A specific temporal order of events at the cellular and molecular leveloccurs in response to injury to the brain. Injury-compromised neuronsdegenerate while surviving neurons undergo neuritogenesis andsynaptogenesis to establish neuronal connectivity destroyed in theinjury. In the brain, after kainate-induced change in neuronal activity(a neurotoxic, excitotoxic or ischemic insult), it was observed that aconsistent down-regulation of neu1 mRNA in the hippocampal formationwith a strong reduction in the molecular layer where granule celldendrites were present. It is suggested, that after injury of the CNS,down-regulation of neu1 mRNA expression leads to reduced levels of Neu1protein that is essential for derepression of the transcription of itstarget genes related to repair and regeneration, such as growth factorsand synaptic proteins.

Neu Role in Memory and Learning

Another function of the Neu family of transcription factors is mediationof Ca²⁺ signaling in a variety of neural processes including learningand memory. A member of Homer family of proteins, Homer2a, is referredto as an interactor of human Neu1 (GenBank Acc. No. AF081530). Homerproteins are enriched in excitatory synapses, bind group I metabotropicglutamate receptors (mGluR), and NMDA receptor interacting Shankproteins and thus can link NMDA and group I mGluR signaling pathways(reviewed in Xiao et al., Curr Opin Neurobiol, 10:370-74 (2000)). Giventhat Homer2a and Neu1 are co-expressed in various brain structures, itis highly conceivable that Neu1 participates in the regulation ofglutamate receptor signalling in the adult brain. Glutamate receptorsignaling has been implicated in several forms of activity-dependentsynaptic plasticity, neurodegenerative diseases, cortical developmentand addiction. Consequently, if Neu is involved in glutamate receptorsignaling, then the change in Neu structure, expression or functioncould lead to various developmental disorders and mental diseases.

Thus, it is highly conceivable that neu1 mRNA expression is regulated byphysiological neuronal synaptic activity leading to reduced levels ofNeu1 protein and derepression of the transcription of its target genesin the processes involving memory and learning. This function of the Neufamily transcription factors makes them a good target for a variety ofdrugs that control different processes in the brain, during developmentand in disease. Manipulating this function of Neu can be used to controla variety of diseases including depression, pain, anxiety, andneurodegenerative diseases.

Neu Role in Tumorigenesis

Also, this invention relates to the role of the Neu family of factors inthe development of tumors, since the human neu1 gene has been mapped tochromosome 10 within a region which is frequently deleted in gliomas.neu1 is expressed at varying levels in different neural andneuroendocrine tumors, including neuroblastomas, carcinoids, non-smallcell lung cancers, and gliomas, suggesting that expression of Neu familyof proteins is applicable as tumor-specific markers in clinical tumordiagnostics. Since over-expression of Neu1 blocks DNA synthesis inneuroblastoma and glioma cells, Neu family of proteins may function alsoas tumor suppressor genes.

Neu Role in Myogenesis and Development of Other Organ Systems.

The described herein, especially in the Examples below, show thatexpression of neu1 and neu2 is found to be high in at late embryonic andadult stages of development, also in developing heart and testes. neu3was found to be widely expressed, with highest levels in immune tissuesspleen and thymus and in lung. Expression of neu4 was detected only inmuscle and heart. Since Neu proteins are expressed at various levels inmany different body systems, the role of Neu family of proteins inseveral developmental pathways is apparent. Studies of neu^(mut) flieshave reported the overproduction of nautilus (nau) expressing cells inembryonic and muscle defects in adult stages of development. Corbin etal., Cell, 67:311-323 (1991); Hartenstein et al., Development,116:1203-1220 (1992). nau is a myogenic bHLH factor that plays a role inthe differentiation of muscle progenitors in Drosophila. Keller et al.,Dev Biol, 181:197-212 (1997); Keller et al., Dev Biol, 202:157-171(1998). Notch signaling has been shown to inhibit MyoD expression andblock myogenesis in mouse. Kuroda et al., J Biol Chem, 274:7238-7244(1999). It is possible that during mammalian myogenesis Neu refinesNotch signaling by regulating expression of nau mammalian homologues(MyoD, myf5, myogenin and MRF4).

Another function of the Neu family of transcription factors is mediationof Ca²⁺ signaling in muscle and other tissues. This function of the Neufamily transcription factors makes them a good target for a variety ofdrugs that control different processes in the muscle, and in thosetissues where Neu is expressed. Manipulating Neu expression and functioncan be used to control a variety of diseases including cancer, andmuscle-degenerative diseases, dystrophinopathies, Brody's disease, andmalignant hyperthermia (the last three are caused by the functionalalterations of Ca(2+) signaling).

Neu Interactions with Neurogenic Genes

Additionally, activity of transcriptional regulators is modulatedthrough interactions with other regulatory factors. Analyses ofinteractions between Drosophila neurogenic loci has revealed that neuappears to act upstream of notch (N), enhancer of split (E(spl)) anddelta (Dl), and downstream of mastermind (mam). Boulianne et al., EMBOJ. 10(10), 2975-2983 (1991). Interaction between neu and E(spl) isobserved in the dominant mutation E(spl)Dl, which is a mutation thatenhances the phenotype of split whereas split is a mutation in the Ngene. Enhancement of the split phenotype increases in the presence ofadditional copies of neu gene, but decreases in heterozygotes for neumutations. Molecular interactions between these Drosophila neurogenicgenes are unknown. Expression of rat SHARP1 gene (Rossner, et al., MolCell Neurosci, 10(3-4):460-475 (1997)), one of the vertebrate homologuesof Drosophila E(spl), is almost identical to the neu1 expression patternduring development and in adult tissues. The similarity in expressionpatterns between neu1 and SHARP1 suggests that products of these genescould reciprocally affect the function of the other as it occurs inDrosophila between neu and E(spl) genes.

Neu Interactions with Proteins Implicated in Nuclear Transport

The nucleocytoplasmic transport of functional molecules is mediatedbidirectionally through the nuclear pore complex (NPC), which spans thedouble membranes of the nuclear envelope. It has recently been shownthat signaling between the nucleus and the cytoplasm plays a key role incoordinating the cellular processes such as the cell cycle and celldifferentiation (Yoneda, Cell Struct Funct 25:205-206. 2000). The weakNLS sequences (HKAVKR (SEQ ID NO: 43), RLKITKK (SEQ ID NO: 44)), thatwere identified in mammalian Neu1 proteins, are indeed, implicated inthe regulated nuclear import of Neu1. Importin-α family members areinvolved in the formation of the NLS receptor complexes that govern theprotein transport to the nucleus. Ullman et al., Cell, 90:967-970(1997); and Izaurralde and Adam, RNA, 4:351-364 (1998). We haveidentified importin-α3 as one of the m-Neu interacting proteins by yeasttwo-hybrid screening.

Neu Interactions with Miz1/GBP/PIAS Family of Proteins

Yeast two hybrid screening revealed that Neu1 interacts with NeuI-1which is a new splice variant of Miz1/PIASX zinc finger transcriptionfactor. Miz1 is a sequence specific DNA binding protein that functionsas a positive-acting transcription factor and interacts directly withhomeobox transcription factor Msx2. Wu et al., 1997 Mech Dev,65(1-2):3-17 (1997). Msx1 and Msx2, members of the Msx family ofhomeobox genes, were found to be important in inductive tissueinteractions. Whereas Msx3 was expressed exclusively in the developingnervous system. Wang, et al., Mech Dev, 58(1-2):203-15 (1996). Membersof the PIAS family, however, regulated DNA binding of STAT transcriptionfactors thereby interfering with the signaling of a variety ofcytokines. Chuang, et al., Biochem Biophys Res Commun, 18;235(2):317-20(1997) and Liu et al., Proc Natl Acad Sci USA, 95(18):10626-31 (1998).

Sequence analyses revealed that PIAS1 is identical to Gu/RNA helicase II(Gu/RH-II) binding protein GBP. Valdez et al., Biochem Biophys ResCommun, 234(2):335-40 (1997). The GBP regulates proteolytic cleavage ofGu/RH-II which could alter its functions or enzymatic activities or leadto its destruction. These data indicate that the Neu family of proteinscould be involved, perhaps through its interactions with variousproteins, such as the NeuI-1 protein. Such interactions may be involvedin several biologically important regulatory processes includinginductive tissue interactions (Miz1), cytokine signaling (PIAS), and RNAprocessing (GBP).

Neu Interactions with ZNF127 Zinc Finger Family of Proteins

Yeast two hybrid screening revealed that Neu1 interacts with NeuI-2.This protein is a new member of the ZNF127 zinc finger family ofproteins. The ZNF127 gene is localized in Angelman/Prader-Willi region.Disruption of this gene causes a genetic defect related to mentalretardation. ZNF127, as well as other genes in this region, weresubjected to genomic imprinting (Mowery-Rushton et al., DNA methylationpatterns in human tissues of uniparental origin using a zinc-finger gene(ZNF127) from the Angelman/Prader-Willi region Am J Med Genet,61(2):140-6 (1996)). The role of ZNF127 in the development ofAngelman/Prader-Willi syndrome, as well as its molecular function, isunknown. The ZNF127 family proteins, however, contain the zinc fingermotif, Cx(8)Cx(5)Cx(3)-H, which is characteristic for viral and earlyimmediate genes such as TIS11, ERF-2 (Tabara et al., 1999 pos-1 encodesa cytoplasmic zinc-finger protein essential for germline specificationin C. elegans Development, 126(1):1-11); and also to RNA bindingproteins (Carballo et al. Science, 281:1001-1005 (1998)).

Neu Interactions with Parkin-Like Proteins

The NeuI-3 protein is another interactor with Neu1 and was found to besimilar to the recently described human gene parkin (Kitada, et al.,Nature 392(6676):605-8 (1998). Mutations in the parkin gene have beenshown to result in autosomal recessive juvenile parkinsonism. Themolecular function of the parkin encoded protein is unknown.Phylogenetic analysis reveals that ari and parkin are distant members ofa common progeny.

Neu Interactions with Androgen Receptor Coregulator ARA54

NeuI-4, another Neu interactor, is identical to ARA54, an androgenreceptor coregulator (Kang et al., J. Biol. Chem. 274, 8570-8576; 1999).Furthermore, the RING-zinc finger domain of NeuI-4 has high similarityto Drosophila protein ariadne (ari) that could be involved in axonalpath-finding. Aguilera, et al., Genetics 155(3):1231-44 (1996). Twomammalian homologues of ari have been identified, however, noinformation is available about molecular mechanisms of the functioningof the ari family of proteins.

Neu Function Based on the Nature of Neu Interactors

Based on the nature of Neu1 interactors, it is hypothesized that Neu hasthe potential to interfere with inductive tissue interactions(NeuI-1/Miz), cytokine signalling (NeuI-1/PIAS), RNA processing(NeuI-1/GBP), early immediate responses (NeuI-2/ZNF127), death ofspecific cell populations (NeuI-3/parkin), and nuclear hormone receptorsignaling and axonal path-finding (NeuI-4/ariadne).

Nucleic Acids

Having identified a number of potential functions for the Neu family ofproteins, the described invention seeks to utilize this knowledge tomanipulate the various developmental pathways in which Neu functions. Asa preliminary step, representative members of the Neu family of proteinshave been isolated and purified. Polynucleotide molecules encoding theproteins of the Neu family were then isolated and their sequences areprovided below.

Representative polynucleotide molecules encoding members of the Neufamily include sequences comprising SEQ. ID. NOs.: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33. Polynucleotide moleculesencoding Neu family members include those sequences resulting in minorgenetic polymorphisms, differences between species, and those thatcontain amino acid substitutions, additions, and/or deletions.

In some instances, one can employ such changes in the sequence of arecombinant Neu to substantially decrease or even increase thebiological activity of Neu relative to the wild-type Neu activity. Suchchanges can also be directed towards endogenous neu sequences using, forexample, gene therapy methods to alter the gene product. Advantageously,the disclosed sequences can be used to identify and isolate neupolynucleotide encoding molecules from suitable vertebrate host cells.Thus, in another embodiment, a method of identifying neu polynucleotidemolecules is provided.

The nucleotide sequences encoding the neuralized homology repeat domaincan be used to identify polynucleotide molecules encoding other proteinsof the Neu family. Complementary DNA molecules encoding Neu familymembers can be obtained by constructing a cDNA library from mRNA of, forexample, brain or muscle tissues that are at different developmentalstages. DNA molecules encoding Neu family members can be isolated fromsuch a library using the disclosed sequences in standard hybridizationtechniques or by amplification of sequences using polymerase chainreaction (PCR) amplification.

In a similar manner, genomic DNA encoding Neu can be obtained usingprobes designed from the sequences disclosed herein. Suitable probes foruse in identifying Neu family sequences can be obtained fromNeu-specific sequences that are highly conserved regions betweenmammalian coding sequences. Primers, for example, from the neuralizedhomology motif domains 1 and 2 are suitable for use in designing PCRprimers. Alternatively, oligonucleotides containing specific DNAsequences from a neu family coding region can be used to identifyrelated human neu genomic and cDNA clones. One of skill in the art willappreciate that upstream regulatory regions of the neu family of genescan be obtained using similar methods.

neu family polynucleotide molecules can be isolated using standardhybridization techniques with probes of at least about 7 nucleotides inlength and up to and including the full coding sequence. Other membersof the neu family can be identified using degenerate oligonucleotidescapable of hybridization based on the sequences disclosed herein for usePCR amplification or by hybridization at moderate or greater stringency.The term, “capable of hybridization” as used herein means that thesubject nucleic acid molecules (whether DNA or RNA) anneal to anoligonucleotide of 15 or more contiguous nucleotides of SEQ. ID. NOs.:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.

The choice of hybridization conditions will be evident to one skilled inthe art and will generally be guided by the purpose of thehybridization, the type of hybridization (DNA-DNA or DNA-RNA), and thelevel of desired relatedness between the sequences. Methods forhybridization are well established in the literature. One of ordinaryskill in the art realizes that the stability of nucleic acid duplexeswill decrease with an increased number and location of mismatched bases;thus, the stringency of hybridization can be used to maximize orminimize the stability of such duplexes. Hybridization stringency can bealtered by: adjusting the temperature of hybridization; adjusting thepercentage of helix-destabilizing agents, such as formamide, in thehybridization mix; and adjusting the temperature and salt concentrationof the wash solutions. In general, the stringency of hybridization isadjusted during the post-hybridization washes by varying the saltconcentration and/or the temperature, resulting in progressively higherstringency conditions.

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and0.1×SSC at about 68° C. (high stringency conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, or each of the conditions can be used, e.g., for 10-15minutes each, in the order listed above, repeating any or all of thesteps listed. However, as mentioned above, optimal conditions will vary,depending on the particular hybridization reaction involved, and can bedetermined empirically. In general, conditions of high stringency areused for the hybridization of the probe of interest.

Alternatively, polynucleotides having substantially the same nucleotidesequence set forth in SEQ. ID. NOs.: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, and 33 or functional fragments thereof, ornucleotide sequences that are substantially identical to SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, canrepresent members of the Neu family of proteins. By “substantially thesame” or “substantially identical” is meant a nucleic acid orpolypeptide exhibiting at least 80%, 85%, 90%, 95% or 100% homology to areference nucleic acid. For nucleotide sequences, the length ofcomparison sequences will generally be at least 10 to 500 nucleotides inlength. More specifically, the length of comparison will be at least 50nucleotides, at least 60 nucleotides, at least 75 nucleotides, and atleast 110 nucleotides in length.

One embodiment of the invention provides isolated and purifiedpolynucleotide molecules encoding Neu proteins, wherein thepolynucleotide molecules that are capable of hybridizing under moderateto stringent conditions to an oligonucleotide of 15 or more contiguousnucleotides of SEQ. ID. NOs.: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, and 33, including complementary strands thereto.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization or computer-basedtechniques which are well known in the art. Such techniques include, butare not limited to: 1) hybridization of genomic or cDNA libraries withprobes to detect homologous nucleotide sequences; 2) antibody screeningof expression libraries to detect cloned DNA fragments with sharedstructural features; 3) polymerase chain reaction (PCR) on genomic DNAor cDNA using primers capable of annealing to the DNA sequence ofinterest; 4) computer searches of sequence databases for similarsequences; and 5) differential screening of a subtracted DNA library.

Screening procedures which rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate probe is available. Oligonucleotide probes, which correspondto a part of the neu family of sequences provided herein and encoding aNeu protein family member, can be synthesized chemically. This requiresthat short, oligo-peptide stretches of the amino acid sequence be known.The DNA sequence encoding the protein can be deduced from the geneticcode, however, the degeneracy of the code must be taken into account. Itis possible to perform a mixed addition reaction when the sequence isdegenerate. This includes a heterogeneous mixture of denatureddouble-stranded DNA. For such screening, hybridization is preferablyperformed on either single-stranded DNA or denatured double-strandedDNA. Hybridization is particularly useful in the detection of cDNAclones derived from sources where an extremely low amount of mRNAsequences relating to the polypeptide of interest are present. In otherwords, by using stringent hybridization conditions directed to avoidnon-specific binding, it is possible, for example, to allow theautoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement. (Wallace, et al., Nucl. Acid Res.,9:879, 1981). Alternatively, a subtractive library is useful forelimination of non-specific cDNA clones.

Among the standard procedures for isolating cDNA sequences of interestis the formation of plasmid- or phage-carrying cDNA libraries which arederived from reverse transcription of mRNA which is abundant in donorcells that have a high level of gene expression. When used incombination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAcan be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).

The nucleotide sequences of the present invention have a myriad ofapplications. Representative uses of the nucleotide sequences of theinvention include the construction of cDNA and oligonucleotide probesuseful in Northern, Southern, and dot-blot assays for identifying andquantifying the level of expression of Neu family proteins in a cell.Lack of expression of a Neu protein in tumors, diseased cells or tissuescan indicate that measuring the level of Neu expression can provideprognostic markers for assessing the growth rate and invasiveness of atumor.

In addition, considering the important role of Neu in development andCa²⁺ signaling, it is thought highly likely that birth defects,degenerative, and psychiatric diseases can result from expression of anabnormal Neu protein. In this case, the Neu protein family can provehighly useful in prenatal screening of mothers and/or for in uterotesting of fetuses. Also, early diagnosis of degenerative andneurological diseases can be based on the analyses of changes in Neuexpression and mutations in the neu genes.

Similarly, the nucleotide sequences can be employed for the constructionof recombinant cell lines, ova, and transgenic embryos and animalsincluding dominant-negative and “knock-out” recombinant cell lines inwhich the regulatory activity of Neu protein is down-regulated oreliminated. Such cells can contain altered Neu coding sequences thatresult in the expression of a Neu protein that is not capable ofenhancing, suppressing or activating transcription of the target gene.The subject cell lines and animals find use in screening for candidatetherapeutic agents capable of either substituting for a functionperformed by Neu or correcting the cellular defect caused by a defectiveNeu.

The Neu family of proteins presents an attractive set of diagnostic andtherapeutic targets, considering the important regulatory role thisfamily of proteins plays in the development function of adult organisms.This important role is reflected by the effects one or defects in amutant Neu protein can inflict upon an organism. Moreover, with theadvances in art of gene therapy progresses, these defects can becorrectable in utero or in early post-natal life or alternativelythrough the use of compounds identified in screening assays using Neuproteins. In addition, neu polynucleotide molecules can be joined toreporter genes, such as beta-galactosidase, luciferase, or greenfluorescent proteins (GFP) and inserted into the genome of a suitablehost cell such as an embryonic or tissue specific stem cell by, forexample, homologous recombination. Cells expressing neu can then beobtained by subjecting the differentiating cells to cell sorting,leading to the purification of a population of neu expressing cells.These cells can be useful for studying specific activity of isolatedcell populations. Also, these cells can be used to study sensitivity togrowth factors or chemotherapeutic agents.

In yet another application of the nucleotide sequence discovery, thetechnology can be useful in the construction of gene transfer vectors(e.g., retroviral vectors, and the like). In these vectors, the neusequence is often inserted into the coding region of the vector underthe control of a promoter. neu gene therapy can be used to correctneurological and movement diseases and cancer. For these therapies, genetransfer vectors can either be injected directly at the site of diseasedcells, or the vectors can be used to construct transformed host cellsthat are then injected at the site of disease.

In one embodiment, a vector comprising a DNA molecule coding a Neuprotein is provided. Preferably, a DNA molecule coding a Neu protein isinserted into a suitable expression vector, which is in turn used totransfect or transform a suitable host cell. Exemplary expressionvectors for use in carrying out the present invention include a promotercapable of directing the transcription of a polynucleotide molecule ofinterest in a host cell. Representative expression vectors include bothplasmid and/or viral vector sequences. Suitable vectors includeretroviral vectors, vaccinia viral vectors, CMV viral vectors,BLUESCRIPT (Stratagene, San Diego, Calif.) vectors, baculovirus vectors,and the like. In another embodiment, promoters capable of directing thetranscription of a cloned gene or cDNA can be inducible or constitutivepromoters and include viral and cellular promoters. In particularlypreferred embodiments, viral vectors are employed for use in expressingNeu proteins in mammalian cells particularly if neu is used for genetherapy.

In some embodiments, it can be preferable to use a selectable marker toidentify cells that contain the cloned DNA. Selectable markers aregenerally introduced into the cells along with the cloned DNA moleculesand include genes that confer resistance to drugs, such as neomycin,hygromycin, and methotrexate. Selectable markers can also complementauxotrophies in the host cell. Other selectable markers providedetectable signals, such as beta-galactbsidase to identify cellscontaining the cloned DNA molecules. Advantageously, the selectablemarkers are amplifiable. Such amplifiable selectable markers can be usedto amplify the number of sequences integrated into the host genome.

Antisense

Antisense neu nucleotide sequences can be used to block expression ofmutant neu expression in a variety of cell types. Suitable antisenseoligonucleotides are at least 11 nucleotides in length and can includeuntranslated (upstream or intron) and associated coding sequences. Aswill be evident to one skilled in the art, the optimal length of an antisense oligonucleotide depends on the strength of the interaction betweenthe antisense oligonucleotide and the complementary mRNA, thetemperature and ionic environment in which translation takes place, thebase sequence of the antisense oligonucleotide, and the presence ofsecondary and tertiary structure in the mRNA and/or in the antisenseoligonucleotide. Suitable target sequences for antisenseoligonucleotides include intron-exon junctions (to prevent propersplicing), regions in which DNA/RNA hybrids will prevent transport ofmRNA from the nucleus to the cytoplasm, initiation factor binding sites,ribosome binding sites, and sites that interfere with ribosomeprogression.

Antisense oligonucleotides can be prepared, for example, by theinsertion of a DNA molecule containing the target DNA sequence into asuitable expression vector such that the DNA molecule is inserteddownstream of a promoter in a reverse orientation as compared to thegene itself. The expression vector can then be transduced, transformedor transfected into a suitable cell resulting in the expression ofantisense oligonucleotides. Alternatively, antisense oligonucleotidescan be synthesized using standard manual or automated synthesistechniques. Synthesized oligonucleotides are introduced into suitablecells by a variety of means including electroporation, calcium phosphateprecipitation, or microinjection. The selection of a suitable antisenseoligonucleotide administration method will be evident to one skilled inthe art. With respect to synthesized oligonucleotides, the stability ofantisense oligonucleotide-mRNA hybrids are advantageously increased bythe addition of stabilizing agents to the oligonucleotide. Stabilizingagents include intercalating agents that are covalently attached toeither or both ends of the oligonucleotide. In preferred embodiments,the oligonucleotides are made resistant to nucleases by, for example,modifications to the phosphodiester backbone by the introduction ofphosphotriesters, phosphonates, phosphorothioates, phosphoroselenoates,phosphoramidates, phosphorodithioates, or morpholino rings.

Protein Production

As would be evident to one skilled in the art, the polynucleotidemolecules of the present invention can be expressed in a variety ofprokaryotic and eucaryotic organisms. For example, the Neu family ofproteins can be expressed in to, Saccharomyces cerevisiae, filamentousfungi, and bacteria, such as E. coli to produce Neu proteins. Similarly,one can express the protein of the described invention in other hostcells such as avian, insect, and plant cells using regulatory sequences,vectors, and methods well established in the literature.

Neu proteins produced according to the present invention can be purifiedusing a number of established methods such as affinity chromatographyusing anti-Neu antibodies coupled to a solid support. Fusion proteins ofantigenic tag and Neu can be purified using antibodies to the tag.Optionally, additional purification is achieved using conventionalpurification means such as liquid chromatography, gradientcentrifugation, and gel electrophoresis, among others. Methods ofprotein purification are known in the art and can be applied to thepurification of recombinant Neu described herein.

Amino Acids

In one embodiment, the identification of mammalian Neu genes isprovided. Preferably, the mammalian Neu genes have highly conservedsequences across the neuralized homology motif domains at the amino acidlevel (Neu1, Neu 2, Neu3, and Neu 4). The following Neu polypeptides orproteins have been identified: human neural Neu1 protein of SEQ. ID.NO.:2, human muscle Neu1 protein SEQ. ID. NO.:4; human Neu1alternatively spliced form (h-neu1ΔNHR1) of SEQ. ID. NO.: 6; mouseneural Neu1 protein of SEQ. ID. NO.: 8; mouse muscle Neu1 protein SEQ.ID. NO.: 10; mouse Neu1 alternatively spliced form (m-neu1ΔNHR2A) ofSEQ. ID. NO.: 12; mouse Neu1 alternatively spliced form (m-neu1ΔNHR2B)of SEQ. ID. NO.: 14; rat Neu 1 protein SEQ. ID. NO.: 16; rat Neu1alternatively spliced form (r-neu1ΔNHR2A) of SEQ. ID. NO.: 18; rat Neu1alternatively spliced form (r-neu1ΔNHR2B) of SEQ. ID. NO.: 20; humanNeu2 protein of SEQ. ID. NO.: 22; human Neu2 alternatively spliced form(h-neu2ΔNHR1) of SEQ. ID. NO.: 24; human Neu2 alternatively spliced form(h-neu2ΔNHR2) of SEQ. ID. NO.: 26; rat Neu 2 protein SEQ. ID. NO.: 28;human Neu3 protein of SEQ. ID. NO.: 30; mouse Neu3 protein of SEQ. ID.NO.: 32; and human Neu4 protein (partial) of SEQ. ID. NO.: 34.

The described invention encompasses Neu variants that, for example, aremodified in a manner that results in Neu proteins capable oftranslocating into the nucleus but unable to repress transcription.Fragments of Neu proteins that are capable of transcriptional repressionbut are incapable of translocating into the nucleus are also encompassedby the present description. Proteins retrieved from naturally occurringmaterials and closely related, functionally similar proteins retrievedby antisera specific to Neu, and recombinantly expressed proteinsencoded by genetic materials (DNA, RNA, cDNA) retrieved on the basis oftheir similarity to the unique regions in the neu family of genes, arealso encompassed by the present description.

According to the present description, polynucleotide molecules encodingNeu encompass those molecules that encode Neu proteins or peptides thatshare identity with the sequences shown in SEQ. ID. NOs.: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34. Such moleculespreferably share greater than 30% identity at the amino acid level withthe disclosed sequences in Neu. In preferred embodiments, thepolynucleotide molecules can share greater identity at the amino acidlevel across highly conserved regions such as the neuralized homologyrepeat domains and the RING-zinc finger domains.

It is contemplated that amino acid sequences substantially the same asthe sequences set forth in SEQ. ID. NOs.: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, and 34, are encompassed by the describedinvention. A preferred embodiment includes polypeptides havingsubstantially the same sequence of amino acids as the amino acidsequence set forth in SEQ ID NOs.: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, and 34, or functional fragments thereof, oramino acid sequences that are substantially identical to SEQ ID NOs.: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34. By“substantially the same” or “substantially identical” is meant apolypeptide exhibiting at least 80%, preferably 85%, more preferably90%, and most preferably 95% homology to a reference amino acidsequence. For polypeptides, the length of comparison sequences willgenerally be at least 16 amino acids, preferably at least 20 aminoacids, more preferably at least 25 amino acids, and most preferably 35amino acids.

Homology is often measured using sequence analysis software (e.g.,Sequence Analysis Software Package of the Genetics Computer Group,University of Wisconsin Biotechnology Center, 1710 University Avenue,Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various substitutions, deletions,substitutions, and other modifications.

The term “functional fragments” include those fragments of SEQ ID NOs.:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, orother Neu family members that retain the function or activity of a Neutranscriptional regulator. One of skill in the art can screen for thefunctionality of a fragment by using the examples provided herein, wherefull-length Neu transcriptional factors are described. It is alsoenvisioned that fragments of various Neu proteins that inhibit orpromote transcription can be identified in a similar manner. Neutranscriptional activity can also be assayed by standard transcriptionassays.

By “substantially identical” is also meant an amino acid sequence whichdiffers only by conservative amino acid substitutions, for example,substitution of one amino acid for another of the same class (e.g.,valine for glycine, arginine for lysine, etc.) or by one or morenon-conservative substitutions, deletions, or insertions located atpositions of the amino acid sequence which do not destroy the functionof the protein assayed, (e.g., as described herein). Preferably, such asequence is at least 85%, more preferably identical at the amino acidlevel to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, and 34.

By a “substantially pure polypeptide” is meant a Neu protein that hasbeen separated from components which naturally accompany it. Typically,the polypeptide is substantially pure when it is at least 60%, byweight, free from the proteins and naturally occurring organic moleculeswith which it is typically associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, Neu protein. A substantially pure Neu polypeptide can beobtained, for example, by extraction from a natural source; byexpression of a recombinant nucleic acid encoding a Neu polypeptide; orby chemically synthesizing the protein. Purity can be measured by anyappropriate method, e.g., column chromatography, polyacrylamide gelelectrophoresis, or by HPLC analysis.

A protein is substantially free of naturally associated components whenit is separated from those contaminants that accompany it in its naturalstate. Thus, a protein that is chemically synthesized or produced in acellular system different from the cell from which it naturallyoriginates will be substantially free from its naturally associatedcomponents. Accordingly, substantially pure polypeptides include thosederived from eukaryotic organisms but synthesized in E. coli or otherprokaryotes.

As would be evident to one skilled in the art, the polynucleotidemolecules of the present disclosure can be expressed in a variety ofprokaryotic and eucaryotic cells using regulatory sequences, vectors,and methods well established in the literature.

Neu proteins produced according to the present invention can be purifiedusing a number of established methods such as affinity chromatographyusing anti-Neu antibodies coupled to a solid support. Fusion proteins ofantigenic tag and Neu can be purified using antibodies to the tag.Optionally, additional purification is achieved using conventionalpurification means such as liquid chromatography, gradientcentrifugation, and gel electrophoresis, among others. Methods ofprotein purification are known in the art and can be applied to thepurification of recombinant Neu described herein.

Construction of interspecies hybrid Neu proteins and hybrid Neu proteinscontaining one or more domains from another Neu family member are alsocontemplated. Such hybrid proteins facilitate structure-functionanalyses. Similarly, hybrid proteins allow for the alteration of Neuactivity by increasing or decreasing the transcriptional regulation oftarget genes. Hybrid proteins of the present invention contain thereplacement of one or more contiguous amino acids of the native Neu withthe analogous amino acid(s) of Neu from another species or other proteinof the Neu family. Such interspecies or interfamily hybrid proteinsinclude hybrids having whole or partial domain replacements. Such hybridproteins are obtained using recombinant DNA techniques well known by oneof skill in the art. Briefly, DNA molecules encoding the hybrid Neuproteins of interest are prepared using generally available methods suchas PCR mutagenesis, site-directed mutagenesis, and/or restrictiondigestion and ligation. The hybrid DNA is then inserted into expressionvectors and introduced into suitable host cells.

One embodiment of the present invention involves the isolation ofproteins that interact with Neu proteins and regulate Neu proteinfunction or are regulated by Neu. Neu proteins can be used inimmunoprecipitation to isolate interacting factors or used for thescreening of interactors using different methods of two hybridscreening. Isolated interactors of Neu can be used to modify Neuactivity or Neu can be used to modify the activity of interactors. Twohybrid screening has resulted in the isolation of several types ofinteractors. Sequence analyses showed that all interactors are novelproteins and contain RING-zinc finger domain located in the C-terminusof the protein. NeuI-1 (4 clones) is a novel splice variant (SEQ. ID.NOS.: 35, 36) of zinc finger protein Miz1/PIASXα/ARIP3 (GenBankaccession numbers NM_(—)008602; AF077953; AF077954; AF044058). NeuI-2 (3clones) is a fourth homolog (SEQ. ID. NOS.: 37, 38; GenBank accessionnumber AF277171; AF302084) of zinc finger protein ZNF127 (GenBankaccession numbers U19106; U19107). NeuI-3 (9 clones) has highesthomology to a human hypothetical protein (GenBank accession numberAK001459) and to a Drosophila hypothetical protein (AAF56052.2) producedfrom CG4813 gene of a genomic scaffold (GenBank accession numberAE003740) (SEQ. ID. NOS.: 39, 40). NeuI-4 (12 clones) is the homolog ofthe androgen receptor coactivator ARA54 (SEQ. ID. NO.: 32; GenBankaccession number AF060544) (SEQ. ID. NO.: 41, 42).

In still another embodiment, synthetic peptides, recombinantly derivedpeptides, fusion proteins, chiral proteins (stereochemical isomers,racemates, enantiomers, and D-isomers) and the like are provided whichinclude a portion of Neu or the entire protein. The subject peptideshave an amino acid sequence encoded by a nucleic acid which hybridizesunder stringent conditions with an oligonucleotide of 15 or morecontiguous nucleotides of SEQ. ID. NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33. Representative amino acid sequences ofthe subject peptides are disclosed in SEQ. ID. NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34. The subject peptidesfind a variety of uses, including preparation of specific antibodies andpreparation of antagonists of Neu activity.

Antibodies

As noted above, the described teachings provide antibodies that bind toNeu. The production of non-human antisera or monoclonal antibodies(e.g., murine, lagomorph, porcine, equine) is well known and can beaccomplished by, for example, immunizing an animal with Neu protein orpeptides. Additionally, catalytic antibodies to nuclear isoforms of theNeu family of proteins or Neu protein metabolic intermediates that aretransported into and out of the nucleus can be generated. For theproduction of monoclonal antibodies, antibody producing cells areobtained from immunized animals, immortalized and screened, or screenedfirst for the production of the antibody that binds to the Neu proteinor peptides and then immortalized. It can be desirable to transfer theantigen binding regions (e.g., F(ab′)2 or hypervariable regions) ofnon-human antibodies into the framework of a human antibody byrecombinant DNA techniques to produce a substantially human molecule.

Following synthesis or expression and isolation or purification of a Neuprotein or a portion thereof, the isolated or purified protein can beused to generate antibodies and tools for identifying agents thatinteract with the Neu protein and fragments of the Neu protein.Depending on the context, the term “antibodies” can encompasspolyclonal, monoclonal, chimeric, single chain, Fab fragments andfragments produced by a Fab expression library. Antibodies thatrecognize Neu proteins and fragments of Neu proteins have many usesincluding, but not limited to, biotechnological applications,therapeutic/prophylactic applications, and diagnostic applications.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, etc. can be immunized by injection with Neuproteins or any portion, fragment or oligopeptide that retainsimmunogenic properties. Depending on the host species, various adjuvantscan be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacteriumparvum are also potentially useful adjuvants.

Peptides used to induce specific antibodies can have an amino acidsequence consisting of at least three amino acids, and preferably atleast 10 to 15 amino acids. Preferably, short stretches of amino acidsencoding fragments of Neu proteins are fused with those of anotherprotein such as keyhole limpet hemocyanin such that an antibody isproduced against the chimeric molecule. While antibodies capable ofspecifically recognizing Neu proteins can be generated by injectingsynthetic 3-mer, 10-mer, and 15-mer peptides that correspond to aprotein sequence of Neu proteins into mice, a more diverse set ofantibodies can be generated by using recombinant Neu proteins, purifiedNeu proteins, or fragments of Neu proteins.

To generate antibodies to Neu proteins and fragments of Neu proteins, asubstantially pure Neu protein or a fragment of Neu protein is isolatedfrom a transfected or transformed cell. The concentration of thepolypeptide in the final preparation is adjusted, for example, byconcentration on an Amicon filter device, to the level of a fewmicrograms/ml. Monoclonal or polyclonal antibody to the polypeptide ofinterest can then be prepared as follows:

Monoclonal antibodies to Neu proteins or a fragment of Neu proteins canbe prepared using any technique that provides for the production ofantibody molecules by continuous cell lines in culture. These include,but are not limited to, the hybridoma technique originally described byKoehler and Milstein (Nature 256:495-497 (1975), the human B-cellhybridoma technique (Kosbor et al. Immunol Today 4:72 (1983); Cote et alProc Natl Acad Sci 80:2026-2030 (1983), and the EBV-hybridoma techniqueCole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc,New York N.Y., pp 77-96 (1985). In addition, techniques developed forthe production of “chimeric antibodies”, the splicing of mouse antibodygenes to human antibody genes to obtain a molecule with appropriateantigen specificity and biological activity can be used. (Morrison etal. Proc Natl Acad Sci 81:6851-6855 (1984); Neuberger et al. Nature312:604-608(1984); Takeda et al. Nature 314:452-454(1985).Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce Neuprotein-specific single chain antibodies. Antibodies can also beproduced by inducing in vivo production in the lymphocyte population orby screening recombinant immunoglobulin libraries or panels of highlyspecific binding reagents as disclosed in Orlandi et al., Proc Natl AcadSci 86: 3833-3837 (1989), and Winter G. and Milstein C; Nature349:293-299 (1991).

Antibody fragments that contain specific binding sites for Neu proteinscan also be generated. For example, such fragments include, but are notlimited to, the F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule and the Fab fragments that can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity. (Huse W. D. et al. Science 256:1275-1281 (1989)).

By one approach, monoclonal antibodies to Neu proteins or fragmentsthereof are made as follows. Briefly, a mouse is repetitively inoculatedwith a few micrograms of the selected protein or peptides derivedtherefrom over a period of a few weeks. The mouse is then sacrificed,and the antibody producing cells of the spleen isolated. The spleencells are fused in the presence of polyethylene glycol with mousemyeloma cells, and the excess unfused cells destroyed by growth of thesystem on selective media comprising aminopterin (HAT media). Thesuccessfully fused cells are diluted and aliquots of the dilution placedin wells of a microtiter plate where growth of the culture is continued.Antibody-producing clones are identified by detection of antibody in thesupernatant fluid of the wells by immunoassay procedures, such as ELISA,as originally described by Engvall, E., Meth. Enzymol. 70:419 (1980),and derivative methods thereof. Selected positive clones can be expandedand their monoclonal antibody product harvested for use. Detailedprocedures for monoclonal antibody production are described in Davis, L.et al. Basic Methods in Molecular Biology Elsevier, New York. Section21-2.

Polyclonal antiserum containing antibodies to heterogenous epitopes of asingle protein can be prepared by immunizing suitable animals with theexpressed protein or peptides derived therefrom described above, whichcan be unmodified or modified to enhance immunogenicity. Effectivepolyclonal antibody production is affected by many factors related bothto the antigen and the host species. For example, small molecules tendto be less immunogenic than others and can require the use of carriersand adjuvant. Also, host animals vary in response to site ofinoculations and dose, with both inadequate or excessive doses ofantigen resulting in low titer antisera. Small doses (ng level) ofantigen administered at multiple intradermal sites appears to be mostreliable. An effective immunization protocol for rabbits can be found inVaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971).

Booster injections can be given at regular intervals, and antiserumharvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony, O. et al., Chap. 19 in: Handbook of ExperimentalImmunology D. Wier (ed) Blackwell (1973). Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12μM). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher, D.,Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman,Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980). Antibodypreparations prepared according to either protocol are useful inquantitative immunoassays that determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively (e.g., in diagnostic embodimentsthat identify the presence of a Neu protein in biological samples). Itis also contemplated that various methods of molecular modeling andrational drug design can be applied to identify additional Neu proteinfamily members, compounds that resemble a Neu protein or fragment orderivative thereof, and molecules that interact with Neu proteins and,thereby modulate their function.

Additional Applications

The Neu family of proteins presents an attractive set of diagnostic andtherapeutic targets, considering the important regulatory role thisfamily of proteins plays in the development function of adult organisms.This important role is reflected by the effects one or more defects in amutant Neu protein can inflict upon an organism. Moreover, with theadvances in the art of gene therapy, these defects can be corrected inutero or in early post-natal life or alternatively through the use ofcompounds identified in screening assays using Neu proteins.

In some instances, cancer cells, or diseased cells or tissue, cancontain a non-functional Neu protein or can contain no Neu protein dueto a genetic mutation or somatic mutations such that these cells fail tostop proliferating and differentiate. For cancers of this type, thecancer cells can be treated in a manner to cause the over-expression ofwild-type Neu protein to force differentiation and cease proliferationof the cancer cells. Accordingly, a method of treating cancer issimilarly provided.

It is also contemplated that because neu family genes control cellproliferation and Ca²⁺ signaling induced transcriptional processes, thatmanipulating Neu expression and function may be useful in controlling avariety of diseases, a few examples of which include depression, pain,anxiety, neurodegenerative diseases, and cancer.

The practice of the described invention is illustrated in the followingnon-limiting examples. The examples are provided below are not intendedto limit the invention in any way.

EXAMPLE 1 Characterization of neu1 Transcripts

A mouse cDNA library of postnatal day (P) 1 brain (Stratagene, SanDiego, Calif.) was screened with a mouse 0.7 kb EST (GenBank #AA5 18339)cDNA clone corresponding to a region ranging from about the translationinitiation codon to the end of the first neuralized homology repeatdomain (NHR1) of h-neu1 (Nakamura et al., 1998).

Sequence analyses, the results of which are shown in FIG. 1, revealedthat the isolated cDNA clones differed in their 5′ regions encodingmuscle- and brain-specific m-Neu1 proteins with different N-termini(FIG. 1). In the figure, “A” shows an amino acid sequence comparison ofDrosophila, human, rat and mouse neu1 proteins. Various domain regionsof the proteins are illustrated. The regions Brain-N, relating to theneural-specific N-terminal region, and Muscle-N, the muscle-specificN-terminal of mammalian neu proteins, are boxed. The NHR1 and NHR2regions, neuralized homology repeat domains 1 and 2; RZD, RING zincfinger domain are underlined. NLS1 and NLS2, nuclear localization signalsequences, are also shown.

RT-PCR analyses of RNA from mouse and rat brain and skeletal muscleresulted in identification of 6 different Neu1 transcripts in bothspecies (FIG. 1): 1) brain- and muscle-specific transcripts encodingNeu1 with the intact NHR2 domain (574 and 557 amino acids,respectively); 2) brain- and muscle-specific transcripts encoding Neu1protein isoforms that lack the region between NHR1 and NHR2, the entireNHR2 region and different parts of the linker region preceding the RZD(Neu1-ΔNHR2A, 342 and 325 amino acids and Neu1-ΔNHR2B, 291 and 274 aminoacids).

Turning again to FIG. 1, the region between asterisks is absent in thesplice isoform neu1-ΔNHR2A; the region between the open circles isabsent in the splice isoform neu1-ΔNHR2B; the pKB consensus sequencebetween NHR1 and NHR2; LRS, two putative leucine rich sequences in theend of both the NHR; and SP, the serine and proline rich repeats betweenthe NHR2 and RZD are boxed. “B” is a schematic representation of thedomain structure of different mouse and rat Neu1 isoforms. Structures ofthe full length Neu1 protein (neu, 574 amino acids) and of two Neu1isoforms (neu-ΔNHR2A, 342 amino acids, neu-ΔNHR2B, 291 amino acids) areshown. Lines indicate to the alternative splicing resulting in the cDNAsencoding neu-ΔNHR2A and neu-ΔNHR2B isoforms. The numbers below theneu-ΔNHR2A and neu-ΔNHR2B correspond to the amino acids of the fulllength Neu1 protein.

Sequence analyses revealed that alternative splicing in the NHR2 and RZDlinker region occurs in-frame and does not affect the intactness of theRING zinc finger structure. Accordingly, both m-neu1 and r-neu1 genesencode protein isoforms with one or two NHRs followed by the C3HC4-typeRZD in the C-terminus. To date no proteins other than Neu1 proteins havebeen identified that contain NHR-like domains and the function of theNHR-like domains has not been previously identified. The RING zincfinger motifs are present in many regulatory proteins and have beenshown to mediate protein-protein interactions. Saurin et al., TrendsBiochem Sci, 21:208-214 (1996).

Like h-Neu, m-Neu1 and r-Neu do not have sequences that are similar tothe Lys-rich nuclear localization signal (NLS) in d-Neu. However, twosmaller clusters of Arg and Lys rich amino acids (HKAVKAR (SEQ ID NO:43) at 80-85 and RLKITKK (SEQ ID NO: 44) at 107-113) are present in theNHR1 of mouse, rat, and human Neu1 that resemble the phosphorylationconsensus sequence of NLS, m-Neu1, r-Neu, and h-Neu proteins, which arerich in Ser and Thr residues. The presence of these regions suggeststhat Neu1 is regulated by phosphorylation. The region between NHR1 andNHR2 contains also a putative protein kinase B/AKT phosphorylation siteRPRSFT (SEQ ID NO: 45) which is similar to the respective consensussequence RXRXXS/T (SEQ ID NO: 46). Datta, et al., Genes and Dev,13:2905-27 (1999).

The region between NHR2 and RZD of m-Neu1, r-Neu, and h-Neu contains twoimperfect repeats of Pro, Ser, and Thr residues with the consensussequence S/TXPXSPXSXPXSPXXXGXXX(X)SD (SEQ ID NO: 47) where X denotes anyamino acid. It is interesting to note that this SP repeat of mammalianNeu1 proteins is not similar to any known protein motifs.

EXAMPLE 2 Developmentally Regulated Expression of neu1 mRNA in the Mouseand Rat Skeletal Muscle and Brain

An RNase protection analyses (RPA) was used to determine the levels ofm-neu1 and r-neu1 mRNAs in the developing and adult brain and non-neuraltissues (FIG. 2).

Total RNA was isolated from the mouse and rat brain regions, which areindicated (A) and (B) respectively in FIG. 2, and non-neural tissues.Levels of neu1 transcripts were analyzed by RNase protection assays. ThecRNA probes used for detection of m-neu1 transcripts were complementaryto a region encoding the muscle-specific (FIG. 2, left panel) or thebrain-specific (FIG. 2, right panel) N-terminus and first half NHR1 ofm-neu1. For r-neu1, the cRNA probe was complementary to a regionencoding the second half of NHR2 up to the stop codon. Specificprotected fragments are indicated on the right of each panel.

Bottom panels of FIG. 2 show the levels of GAPDH mRNA in the RNAsamples. The nomenclature used to identify each sample is as follows:neu-M, indicates a muscle-specific m-neu1 transcript; neuB, indicates abrain-specific m-neu1 transcript; neu-B/M indicates the total pool ofmuscle- and brain-specific r-neu1 transcripts. Further, E, denotes“Embryonic day”; P, denotes “postnatal day”; ad, denotes “adult”; th,denotes “thymus”; he, denotes “heart”; lu, denotes “lung”; sp, denotes“spleen”; ki, denotes “kidney”; li, denotes “liver”; mu, denotes“skeletal muscle”; gu, denotes “gut”; plac, denotes “placenta”; ctx,denotes “cerebral cortex”; cbl, denotes “cerebellum”; hc, denotes“hippocampus”; str, denotes “striatum”; mid, denotes “ventral midbrain”;hth, denotes “hypothalamus”; col, denotes “colliculi”; thal, denotes“thalamus”; pons, denotes “pons”; olf; denotes “olfactory bulb”; med,denotes “medulla”; pit, denotes “pituitary”; and tRNA, denotes “yeasttRNA”, which was used as a negative control.

The overall highest expression levels of neu1 mRNA were seen in theadult skeletal muscle and brain. In the skeletal muscle, neu1 mRNAlevels were undetectable during embryonic development, low at birth andupregulated during postnatal development reaching the highest levels inthe adult. Other non-neural tissues (heart, kidney, liver, lung, thymus,and spleen) except for the adult heart and testis did not express neu1transcripts or the levels were below the detection limit of the RPA. Inthe brain, low levels of neu1 mRNA were detected at embryonic day (E) 13and the expression increased progressively reaching the highest levelsin the adult. neu1 expression levels were high in the cerebral cortex,hippocampus, and striatum and substantially lower in the olfactorysystem, thalamus/hypothalamus, midbrain, cerebellum, pons, and medulla.

neu1 expression levels were specifically analyzed during postnataldevelopment of cerebral cortex and cerebellum. In both of these brainregions, low levels of neu1 mRNA were observed in postnatal day (P) 1whereas by two weeks after birth the expression levels increasedsignificantly reaching the peak levels in the adult animal. High levelsof neu1 mRNA expression were also detected in the adult dorsal rootganglia and moderate levels in the adult spinal cord. In all the tissuespredominant neu1 transcripts contained intact NHR2 domain, whereas thelevels the neu1 mRNAs lacking the NHR2 (neu1-ΔNHR1A and neu1-ΔNHR1B)were below 5% of all m-neu1 transcripts. Presented data show that neu1is highly expressed during mouse and rat postnatal development and thatthe expression is confined to skeletal muscle and the nervous system.

EXAMPLE 3 Neuronal Expression and Dendritic Localization of MammalianNeuralized mRNA

In situ hybridization analysis was used to study the cellularlocalization of neu1 mRNA expression in the embryonic and adult mousebrain. The results are presented in FIG. 3.

Shown are dark-field emulsion autoradiographs obtained afterhybridization of coronal sections of mouse brain with the [³⁵S]-labeledm-neu1 cRNA probe corresponding to the first neuralized homology region(NHR1) of neu1. m-neu1 mRNA-specific labeling is shown at embryonic day17 (E17, upper panel), at postnatal day 7 (P7, middle panel) and adult(AD, lower panel) in different brain structures. Exposure time was 3weeks for adult and P7 sections and 6 weeks for E17 sections. As shownin the figure, BG, denotes “basal ganglia”; Cx, denotes “cerebralcortex”; Th, denotes “thalamus”; PTec, denotes “pretectum”; SC, denotes“superior colliculi”; IC, denotes “inferior colliculi”; Cb, denotes“cerebellum”; Me, denotes “medulla”; Pit, denotes “pituitary”; P,denotes “pons”; Hc, denotes “hippocampus”; Am, denotes “amygdala”; andPn, denotes “Pontine nuclei”.

Results of these analyses supported the conclusions drawn from the RPAstudies discussed in Example 2. Neuralized1 mRNA-specific labeling wasnot detected at E13 and E15 brain, because of very low expressionlevels. This result was supported by the related data produced from theRPA. At E17, all brain regions, except for the cerebral cortex,hippocampus, and cerebellum, were found to express low levels of m-neu1mRNA. In the brain, m-neu1 expression was confined to the regionscontaining postmitotic neurons and was not present in ventricular andsubventricular zones that contain proliferating neural stem andprogenitor cells.

At P7, the levels of neu1 mRNA increased significantly in the basalganglia, amygdala, hypothalamus, and hippocampus. Low levels werepresent in the cerebral cortex and brainstem. In the adult, widespreadexpression of m-neu1 mRNA was observed throughout the brain particularlyin the cerebral cortex, hippocampal formation, the basal ganglia,amygdaloid, hypothalamus, and pontine nuclei and in the cerebellum,while lower levels were seen in the mesencephalon and medulla oblangata.m-neu1 mRNA-specific signal was not detected in most of the thalamicnuclei.

A more detailed analysis of neu1 expression was carried out in rat brainand the data is shown in Table 2 and in FIGS. 4, 5, and 6.

FIG. 4 shows the results of an in situ hybridization analysis of neu1mRNA expression in adult rat brain. The coronal sections (A-H)correspond to the levels from 5 Bregma in the atlas of Paxinos andWatson (Paxinus and Watson, 1986) as indicated by numbers in the leftpart of the bottom of each autoradiograph. CTX, denotes “cerebralcortex”; CPu, denotes “caudate putamen”; Pir, denotes “piriform cortex”;LS, denotes “lateral septum”; HDB, denotes “nucleus of the horizontallimb of the diagonal band of Broca”; VDB, denotes “nucleus of thevertical limb of the diagonal band of Broca”; GP, denotes “globuspallidus”, BSTM and BSTL, denotes “bed nucleus of the stria terminalis,medial and lateral division respectively”; MHb, denotes “medialhabenular nucleus”; Rt, denotes “reticular thalamic nucleus”; AH,denotes “anterior hypothalamic area”; Zi, denotes “zona incerta”; Am,denotes “amygdaloid nuclei”; VMH, denotes “ventromedial hypothalamicnucleus”; DMD, denotes “dorsomedial hypothalamic nucleus”; DG, denotes“dentate gyrus”; CA1 and CA3, denotes “pyramidal layers of thehippocampus”; Ent, denotes “entorhinal cortex”; PAG, denotes“periaqueductal gray”; Sc, denotes “superior colliculus”; S, denotes“subiculum”; Pn, denotes “pontine nuclei”; GL, denotes “granular layerof cerebellum”; PL, denotes “Purkinje cell layer of cerebellum”; ECu,denotes “external cuneate nucleus”; Ve, denotes “vestibular nuclei”; Gi,denotes “gigantocellular reticular nucleus”; Sp5, denotes “spinaltrigeminal nucleus”: PCRt and IRt, denote “parvicellular reticularnuclei” and “intermedial reticular nuclei”, respectively; LPGi, denotes“lateral paragigantocellular nucleus”; LRt, denotes “lateral reticularnucleus”; and Sol, denotes “solitary nucleus”. The scale bar correspondsto 5 mm in length.

FIG. 5 shows expression of neu1 mRNA in an adult rat nervous system.Dark-field in situ hybridization autoradiographs showing labelling in A,the cerebral cortex; B, CA1-CA3, hippocampal subfields, and in thedentate gyrus (DG) including the strata molecular (Mol), lacunosummolecular (Lmol) and radiatum (Rad); C, substantia nigra compact part(SNc) and reticular part (SNr); D, medial habenular nucleus (MHb); E,locus coeruleus (LC); F, in the molecular (Mol) and granular (Gr) layerof cerebellar cortex; G, in the layers of the retina: ganglion celllayer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL),outer plexiform layer (OPL), outer nuclear layer (ONL), pigmentalepitelial (PE); H, dorsal root ganglia (DRG). Roman numbers in Aindicate the various layers of cerebral cortex; CC, corpus callosum;Hil, hilus; WM, white matter. The scale bars correspond lengths of 200μm in A and 400 μm in B-H.

FIG. 6 shows cellular localization of neu1 mRNA in an adult rat nervoussystem. Bright-field in situ hybridization microphotographs showinglabelled cells in A, in the layer II of the cerebral cortex at the levelof the somatosensory cortex (CTX); B, dentate gyrus (DG) of hippocampus;C, CA3 layer of the hippocampus; D, polymorph layer of dentate gyrus(PoDG); E, lateral septum (LS); F, substantia nigra compact part (SNc);G, globus pallidus (GP); H and I, spinal cord at L3 level, respectivelyin the layers 9 and 2-3 (Sc); J, dorsal root ganglia (DRG); K, glialimitans (GL); L, pineal gland (Pi). Note in G, the cells in the globuspallidus (GP) do not express neu1 mRNA. Mol, stratum moleculare; Hil,hilus of the hippocampus; Rad, stratum radiatum; Or, stratum oriens. Thescale bar corresponds to 25 μm in length.

Overall, although the neu1-specific labeling was distributed broadlythroughout the brain, more predominant expression was confined toforebrain structures and the levels were lower in caudal regions of thebrain, with the exception of some nuclei (Table 2). In the olfactorysystem the anterior olfactory nucleus showed neu1-specific signal. Inthe cerebral cortex neu1 mRNA expression levels were particularly high(FIG. 4A-F, 5A, 6A). In the cerebral cortex, highest levels of neu1 mRNAexpression were found in layer I-III, whereas expression graduallydecreases from layer IV to layer VI. Dense labeling was seen in layersI-III, and in scattered neurons of layer V, moderate levels in layers Vand VI, and low levels in layer IV. (FIG. 5A, 6A) Interestingly,labeling in layer I did not cover diffuse cell bodies, suggestingdendritic localization of neu1 mRNA. Piriform and entorhinal corticieswere also labeled with neu1-specific signal.

In the hippocampus, neurons of the granular layer of dentate gyrus andthe pyramidal layers CA1-CA3 showed moderate-high labeling (FIG. 4C-E,5B, 6B-D). Neurons of the hilus and subiculum expressed low levels ofneu1 mRNA. The most interesting observation regarding neu1 mRNAlocalization in the adult brain was the clear dendritic localization ofthe transcripts in the hippocampus. The neu1-specific labeling over themolecular layer of the dentate granule cells was uniformly distributed(FIG. 4C-E). Examination of emulsion-dipped sections showed that thislabeling did not cover cell bodies and extended up to the hippocampalfissure, showing that neu1 mRNA is localized throughout the entiredendritic tree of dentate granule cell layer (FIG. 5B, 6B). The strataoriens and radiatum of CA1-CA3 of hippocampus, corresponding to thedendrites of these regions, were also labeled by neu1 cRNA but thelevels were significantly lower than in the molecular layer of thedentate gyrus (FIG. 4C-E, 5B, 6C).

In the basal ganglia, r-neu1 cRNA was detected in caudate putamen andaccumbens but not in the globus pallidus (FIG. 4A, B, 6G). Moderate tohigh levels of neu1 mRNA were seen in all the amygdaloid nuclei (FIG.4C-D), the interstitial nucleus of the posterior limb of anteriorcommissure and the bed of stria terminalis of extended amygdala (FIG.4B). In the septum, high levels of labeling were seen in the cells oflateral septum (FIG. 4A; 6E), and very low levels in the vertical andhorizontal limb of the diagonal band of Broca. In the hypothalamus, thesupraoptic, suprachiasmatic, supramammilary, and ventromedial nucleiexpressed moderate levels, and several other nuclei expressed low levelsof neu1 mRNA (FIG. 4B-E). In the thalamus, the medial habenula expressedhigh levels of neu1 mRNA, while moderate levels were observed in thereticular thalamic nucleus and low levels in a few other nuclei of thethalamus, such as the zona incerta, lateral habenula, mediodorsalthalamic nuclei, laterodorsal thalamic nuclei, paraventricular thalamicnucleus, rheuniens, rhomboid thalamic nuclei, and medial pretectalnucleus (Table 1, FIG. 4C-D, 5D). In the mesencephalon, the neurons ofthe substantia nigra, colliculi superior, ventral tegmental area,periaqueductal gray, interpeduncular nucleus, oculomotor nucleus, andRaphe nucleus showed low levels of neu1-specific signal (FIG. 4F, 5C,6F). The granule cell layer of the cerebellum was strongly labeled, butthe Purkinje cells and deep cerebellar nuclei were not (FIG. 4G, H, 5F).Within myelenchepalon, the locus coeruleus, and the pontine, trapezoidbody, facialis, pontine reticular, and rostroventrolateral reticularnuclei were labeled with moderate density with r-neu1 cRNA (Table 1,FIG. 4F, 5E). Beyond this, there was low-density labeling in thetrigeminal, facial, solitary, and hypoglossal nuclei (FIG. 4G, H). Inaddition, neu1 cRNA labeling was evident at low levels or in associationwith fewer cells in several other nuclei, such as the vestibular,cochlear, paragigantocellular, and abducens nuclei (Table 1).

In the retina, labeling of neu1 mRNA was strong in the inner nuclearlayer, with the highest intensity in the outer border. Diffuse labelingwas also seen in the ganglion cell layer and both in the inner and outerplexiform layers (FIG. 5G). Cells in the pineal gland expressed highlevels of neu1 mRNA (FIG. 6L). Finally, labeling was relatively low inthe cells of the spinal cord (FIG. 6H, I), in contrast to the neurons ofdorsal root ganglia, which expressed high levels of neu1 mRNA (FIG. 5H,6J).

At the cellular level, m-neu1 mRNA expression was predominant in cellsdisplaying neuronal profile (large cells with weakly stained nuclei) andnot in the cells with glial profile (small cells with strongly stainednuclei). In addition, neu1 mRNA expression was not detected in the whitematter, where neuronal cells are absent. One of the few exceptions ofnon-neuronal neu1 mRNA expression was glia limitans that displayed denselabeling (FIG. 6K).

Using in situ hybridization in combination with immunocytochemistry forNeuN, a Neuron-specific protein, we found that clusters of silvergrains, reflecting the presence of neu1 mRNA, were located over theperikarya of NeuN-positive cells in all the brain regions examined, suchas cerebral cortex, hippocampal formation, striatum, hypothalamusnuclei, amygdaloid complex, and cerebellum. FIG. 7 shows the results ofneuronal expression studies of neu1 mRNA levels in adult rat brain Inthe figure, neu1 mRNA was visualized as autoradiographic grains whereasNeuN was shown as peroxidase staining resulting in a yellow color. Thetwo labelings co-localized with all the cells. As shown in the figure:A, layer II of cerebral cortex (CTX); B, CA1 layer of the hippocampus;C, polymorph layer of the dentate gyrus (PoDG) and CA3 layer of thehippocampus; D, caudate putamen (CPu); E, reticular thalamic nucleus(Rt); and F, ventromedial hypothalamic nucleus (VMH), were studied Notethat neurons (NeuN-positive cells) of the ventral posterolateralthalamic nucleus (VPL) in E do not express neu1 mRNA.

By evaluation the proportion of cells positive for both neu1 mRNA andNeuN out of the total number of NeuN-containing neurons, it was foundthat all the neurons in the layers II and III of the cerebral cortexexpressed neu1 mRNA. In the layer IV eighty percent and layers V-VIabout ninety percent of neurons expressed neu1 mRNA. In several otherbrain regions examined, such as hippocampal formation, striatum,reticular thalamic nucleus, hypothalamic nuclei, showing that themajority of the neuronal cells express neu1 mRNA. In contrast, neuronsin the globus pallidus and in several thalamic nuclei, such as ventralposterolateral and posteromedial nuclei, did not express neu1 mRNA (FIG.7).

Neuronal activity has been shown to regulate the dendritic localizationof the immediate-early gene arc mRNA. Steward et al., Neuron, 21:741-51(1998); Guzovsky et al., Nat Neurosci, 2:1120-1124 (1999). To examine ifneu1 mRNA dendritic localization is regulated by neuronal activity, theeffect of kainate-induced seizures on the expression and localization ofneu1 mRNA was studied (FIG. 8). At 24 hours after the injection ofkainate, neu1 mRNA levels decreased in all the regions of thehippocampus. The levels of neu1 mRNA are significantly downregulated inthe granular and molecular layers of dentate gyrus of adult rat brain at24 hours after the treatment with kainic acid (B and B1), as compared tocontrol brains (A and A1). Regions analyzed are denoted as follows: CTX,cerebral cortex; DG, dentate gyrus; Am, amygdala; Pir, piriform cortex;VMH, ventromedial hypothalamic nucleus; and Mol, for the molecular layerof the hippocampus. Particularly pronounced was the reduction oflabeling in the molecular layer of the dentate gyrus. Similarly, butless consistently, a down-regulation of neu1 mRNA was observed in thecerebral cortex.

EXAMPLE 4 Neu1 Protein Isoforms Exhibit Transcriptional RepressorActivities

The ability of m-Neu1 to act as a transcriptional regulator was studiedusing a chloramphenicol acetyl-transferase (CAT) assay. Severaltranscriptional regulators, such as the polycomb group-relatedtranscriptional regulator MEL18, the MDM2 proto-oncogene, breast andovarian cancer susceptibility gene BRCA1 and MAT1, a subunit of TFIIHbasal complex factor contain RING finger motifs. Kanno et al., EMBO J,14:5672-5678 (1995); Leveillard and Wasylyk, J Biol Chem,272:30651-30661 (1997); Pao et al., Proc Natl Acad Sci USA, 97:1020-1025(2000); and Fesquet et al., Oncogene, 15:1303-1307 (1997). Furthermore,the earlier studies proposed d-Neu to function as a DNA-bindingtranscription factor. Boulianne et al., EMBO J, 10:2975-2983 (1991) andPrice et al., EMBO J, 12:2411-2418 (1993).

The effect of m-Neu1 on transcriptional activity was studied using achloramphenicol acetyl-transferase (CAT) assay and the followingpromoters: 1) TATA-box containing: 1.0 kb BDNF-I-CAT, 0.3 kbBDNF-II-CAT, 0,7 kb BDNF-IV CAT, 0.4 kb NF-L, 1.0 kb GAP-43, 2)TATA-less promoters with putative initiators (Inr): 0.4 kb BDNF-III-CAT,0.4 kb LNGFR-CAT, and 0.3 kb ME1-CAT. Timmusk et al., Neuron, 10:475-489(1993); Reeben et al., J Neurosci Res, 40:177-188 (1995); Chiaramello etal., J Biol Chem, 271:22035-22043 (1996); Metsis et al., Gene,121:247-254 (1992); and Shain et al., Nucleic Acids Res, 23:1696-1703(1995). Data from the study is shown in FIG. 9.

FIG. 9 show that Neu1 represses transcription from various promoters intransient expression assays. The data for this figure was gathered fromNeuro2A cells that were co-transfected with 0.5 μg of various reporterplasmids containing different promoters driving the epression of CAT andpRcCMV expression plasmid (1.0 μg) without any cDNA sequence (control)or containing m-neu1 cDNAs encoding the full length protein (neu-FL) orthe m-Neu-1-ΔNHR2A (neuΔNHR2A) isoform lacking the region between NHR1and RZD, using FuGENE-6 transfection system. The pON260 expressionplasmid (0.1 μg) encoding β-galactosidase was included in thetransfections to normalize the transfection efficiencies. The CATactivity is defined as 100 for each reporter when cotransfected with thepRcCMV parental expression plasmid. When cotransfected with expressionplasmids containing m-neu1 cDNAs, the CAT activities are expressedrelative to the value obtained by cotransfection of each reporterplasmid and the parental pRcCMV expression plasmid. The data shown arerepresentative of at least three independent experiments. Error barsrepresent the S.E. BDNF-I, 1.0 kb BDNF promoter I, BDNF II, 0.3 kb BDNFpromoter II; BDNF III, 0.4 kb BDNF promoter III; BDNF IV, 0.7 kb BDNFpromoter IV; NF-L, 0.4 kb NF-L promoter; GAP-43, 1.0 kb GAP-43-promoter,LNGFR, 0.4 kb LNGFR promoter; ME1, 0.3 kb ME1 promoter.

The full-length and the isoform of m-Neu1-ΔNHR2A significantly reducedthe activity of all of these promoters in Neuro-2A cells (FIG. 9). CATassays performed in other cell types (mouse teratocarcinoma PCC7, ratastrocytoma C6, human breast ductal carcinoma BT-549, human primaryosteogenic sarcoma Saos2, human cervix carcinoma C-33A) or using adifferent reporter (RARβ promoter driven lac Z) gave similar results(data not shown), suggesting that the transcriptional repressor activityof m-Neu1 does not depend on the reporter and cellular context.

Following the identification of m-Neu1's activity as a transcriptionalregulator, identification of the repression domains of m-Neu1 wasperformed. Expression plasmids were generated encoding full-lengthm-Neu1 or individual domains of m-Neu1 fused to Gal4 DNA-binding domainand tested their activities by CAT assays. The data is presented in FIG.10.

FIG. 10 shows that neuralized homology repeat domains of Neu1 mediatethe transcriptional repression when fused to the DNA binding domain ofGal4. Neuro2A cells were cotransfected with Gal4TK-CAT reporter plasmidcontaining five Gal4 binding sites in front of TK promoter driving theexpression of CAT (0.5 μg) and pBIND expression plasmid (1.0 μg) withoutany cDNA sequence (G4) or containing different regions of m-neu1 cDNAfused in-frame to Gal4 DNA-binding domain using FuGENE-6 transfectionsystem. The designations of the constructs are shown on the left of FIG.10. The CAT activities are expressed relative to the value obtained bycotransfection of the reporter plasmid and the parental pRcCMVexpression plasmid which was set 100. The data shown are representativeof at least three independent experiments. Error bars represent the S.E.

The co-transfection of full-length m-Neu1 fused to Gal4 (Gal4-m-Neu1)with the Gal4-TK-CAT resulted in a concentration-dependent repression ofthe CAT activity (FIG. 10). m-Neu1 isoforms that lack the NHR2 and theregion preceding the RZD also displayed transcriptional repressoractivities when fused to Gal4 DBD (FIG. 10). Extensive deletion analysesshowed that NHR1 and NHR2 domains possess transcriptional repressoractivity, whereas neural and muscle-specific N-termini (amino acids1-60), RZD (amino acids 509-574) and the serine-proline rich linkerregion joining RZD to NHR2 (amino acids 439-510) did not affecttranscription (FIG. 10). Interestingly, individual NHR1 and NHR2displayed even stronger repressor activities than the full-lengthm-Neu1. These data show that m-Neu1 acts as a transcriptional repressorwhen tethered to a promoter via a heterologous DNA binding domain (DBD).To summarize, m-Neu1 represses the activity of both TATA and TATA-lesspromoters in transient expression assays.

EXAMPLE 5 Nucleocytoplasmic Shuttling of Neu1 Protein

The function of Neu1 as a transcriptional repressor implies its nuclearlocalization. A putative lysine rich nuclear localization signal (NLS)that is present in d-Neu protein (d-NLS) is not conserved in mammals.Boulianne et al., EMBO J, 10:2975-2983 (1991) and Price et al., EMBO J,12:2411-2418 (1993). To study the subcellular localization of m-Neu1protein expression constructs were generated encoding taggedm-Neu-fusion proteins. The expression of these vectors was analyzed inNeuro2A cells.

The results from this work is shown in FIG. 11. This figure shows thesubcellular localization of m-Neu1-FLAG and m-Neu1-EGFP fusion proteinsin Neuro2A cells. Neuro2A cells were transfected expression plasmidsencoding m-Neu1-FLAG (A, E, F) and different m-Neu1-EGFP (B, C, D)fusion proteins or with parental pFLAG (G) and pEGFP-C3 (H) expressionplasmids. Numbers indicate the amino acids of Neu1 fused to theC-terminal of FLAG and EGFP. Presented are images of FLAGimmunofluorescence using anti-FLAG antibody (A, E, F, G) and directfluorescence of EGFP fusion proteins (B, C, D, H).

More specifically, A, shows the Neu1-FLAG fusion protein is localizedeither in the cytoplasm or in the nucleus; B, shows that Neu1/120-EGFPfusion protein containing amino acids 1-120 of Neu1 exhibitsconstitutive nuclear localization in all cells; C, shows that theNeu1-60/120-EGFP fusion protein is seen exclusively in the nucleusrevealing that the first 60 amino acids of NHR1 are sufficient fornuclear localization of Neu1; in D, the Neu1-1/60-EGFP fusion proteincontaining the N-terminal 60 amino acids of the brain- specific isoformof m-Neu1 shows localization that is identical to H, localization ofEGFP synthesized from the parental pEGFP-C3 expression plasmid; E,inhibition of nuclear export of Neu1-FLAG fusion protein by treatment ofNeuro2A cells with leptomycin B (10 ng/ml) for 12 hours or F, 2 days ofRA and dBcAMP mediated differentiation leads to predominant nuclearlocalization of Neu1-FLAG fusion protein; G, FLAG synthesized fromparental plasmid is distributed evenly in the cell.

The fusion protein m-Neu1-FLAG comprised of the full-length m-Neu1 withN-terminal FLAG tag showed either predominantly nuclear (˜40% of thepositive cells) or predominantly cytoplasmic (˜60% of the positivecells) distribution. If localized in the cytoplasm, m-Neu1-FLAG fusionprotein was observed in the form of granular speckles in the perinucleararea, in the vicinity of plasma membrane and in neurites (FIG. 11A).

m-Neu1 does not have sequences that are similar to the lysine-richd-NLS, however, two smaller clusters of arginine and lysine rich aminoacids (HKAVKAR (SEQ ID NO: 43) at 80-85 and RLKITKK (SEQ ID NO: 44) at107-113) are present in the NHR1 of m-Neu1 (FIG. 1). To investigatewhether these putative NLSs are functional, the subcellular localizationof m-Neu1 deletion mutants fused to the C-terminus of EGFP were analyzedin living cells. Neu-1/120-EGFP fusion protein containing the first 120amino acids of m-Neu1 displayed nuclear localization in virtually allthe cells (100%) (FIG. 11B). Deletion of the first 60 amino acids fromthe N-terminal region of m-Neu1 up to the NHR1 domain (Neu-60/120-EGFP)did not change the predominant nuclear localization of theNeu-1/120-EGFP fusion protein (FIG. 11C). The N-terminal region of Neu1fused to EGFP (Neu1/120-EGFP) displayed overall cellular distribution ofthe fusion protein (FIG. 11D) which was identical to the localization ofEGFP (FIG. 11H). These results showed that the region containing thefirst 60 amino acids of NHR1 (amino acids 60-120) including two putativeNLS domains are sufficient for nuclear import of m-Neu1.

Localization of m-Neu1 fusion proteins in the nucleus and cytoplasmsuggest that m-Neu1 transport could be the subject of regulation.Various proteins have been shown to be exported from the nucleus by theCRM1/exportin1-related export pathway that is blocked by the antibioticleptomycin B (LMB), a specific inhibitor of nuclear export mediated byleucine-rich nuclear export signals (NES). Hood and Silver, Curr OpinCell Biol, 11:241-247 (1999); Izaurralde and Adam, RNA, 4(:351-364(1998); Ullman et al., Cell, 90:967-970 (1997); Weiss (1998); Fornerodet al., Cell, 90:1051-1060 (1997); Fukuda et al., Nature, 390:308-311(1997); Kudo et al., Exp Cell Res, 242:540-547 (1998); Kudo et al., ProcNatl Acad Sci USA, 96:9112-9117 (1999); and Nishi et al., J Biol Chem,269:6320-6324 (1994). m-Neu1 protein contains two putative leucine-richsequences, one in the end of each of the NHR (FIG. 1), that are similarto the identified NES in different proteins. Kogerman et al., Nat CellBio, 1:312-319 (1999); Taagepera et al., Proc Natl Acad Sci USA,95:7457-7462 (1998); Ullman et al., Cell, 90:967-970 (1997); and Yamagaet al., J Biol Chem, 274:28537-28541 (1999). In view of these results,the effect of LMB on the localization of m-Neu-FLAG in Neuro2A cells wasnext studied.

Treatment of Neuro2A cells transfected with m-Neu1-FLAG expressionconstruct with LMB (10 ng/ml) resulted in exclusively nuclearlocalization of m-Neu1-FLAG at 12 hours post-treatment in virtually allthe cells (FIG. 11E). These results suggest that m-Neu1 protein shuttlesbetween nucleus and cytoplasm, and that the CRM1/exportin1-relatedpathway is involved in nuclear export.

Interestingly, the number of cells with nuclear localization ofm-Neu1-tagged protein increased substantially (from 45% to 80%) 12 hsafter the RA- and cAMP-mediated neuronal differentiation (FIG. 11F). Itsuggests that neuronal differentiation changes the mechanisms that areresponsible for the translocation of m-Neu1 protein in Neuro2A cells.

EXAMPLE 6 Characterization of Neu1 Homologs Neu2, Neu3 and Neu4

Screening of cDNA libraries and RT-PCR amplification has resulted inisolation of several homologs of mammalian neu1 genes. Polynucleotidesequences of the following homologs are presented as SEQ. ID. NOS.:human neu2 cDNA of SEQ. ID. NO.:21; human neu2 alternatively splicedform h-neu2-ΔNHR1 of SEQ. ID. NO.:23; human neu2 alternatively splicedform h-neu2-ΔNHR2 of SEQ. ID. NO.:25; rat neu2 cDNA SEQ. ID. NO.: 27;human neu3 cDNA of SEQ. ID. NO.: 29; mouse neu3 cDNA of SEQ. ID. NO.:31; and human neu4 cDNA (partial) of SEQ. ID. NO.: 33. Correspondingpeptide sequences are presented as SEQ. ID. NOs: human Neu2 protein ofSEQ. ID. NO.: 22; human Neu2 alternatively spliced form h-neu2-ΔNHR1 ofSEQ. ID. NO.:24;; human Neu2 alternatively spliced form h-neu2-ΔNHR2 ofSEQ. ID. NO.:26; rat Neu2 protein SEQ. ID. NO.: 28; human Neu3 proteinof SEQ. ID. NO.: 30; mouse Neu3 protein of SEQ. ID. NO.: 32; and humanNeu4 protein (partial) of SEQ. ID. NO.: 34. All the mammalian Neuproteins show significant homology in the NHR and Ring zinc fingerdomains.

Alignment of neuralized homology motifs of human Neu1, Neu2, and Neu3proteins is shown in FIG. 12. Amino acid sequence comparison of theneuralized homology regions (NHR) of Drosophila neu and human neu1, neu2and neu3 proteins is shown in FIG. 12. Amino acids that are identical inhuman and Drosophila neu proteins are highlighted in white on blackbackground. Amino acids that are similar in human and Drosophila neuproteins are boxed and highlighted on grey background. h1I, NHR1 ofhuman neu1; h2I, NHR1 of human neu2; h3, NHR of human neu3; dI, NHR1 ofDrosophila neu; dII, NHR2 of d-neu; h1II, NHR2 of human neu1; h2II, NHR2of human neu2.

Expression of neu1 mRNA is highest in adult, mature neurons (FIG. 2-7).neu2, in contrast to neu1, is expressed at high levels already in theembryonic brain and the expression levels decrease during postnataldevelopment (FIG. 13). Total RNA was isolated from the indicated mouseand rat brain regions and non-neural tissues and the levels of neu2transcripts were analyzed by RNase protection assays. cRNA probes thatwere used for detection of neu2 transcripts covered the region encodingthe NHR1 of rat neu2. Specific protected fragments are indicated on theleft of the panel. R-NEU2, rat neu2 transcript; M-NEU2, mouse neu2transcript. E, Embryonic day; P, postnatal day; ad, adult; HC,hippocampus; CTX, cerebral cortex; OLF, olfactory bulb; STR, striatum;THA, thalamus; HTH, hypothalamus; COL, colliculi; MID, ventral midbrain;CBL, cerebellum; PONS, pons; MED, medulla;SP.C, spinalcord; PIT,pituitary; MUS, muscle; SPLCE, spleen tRNA, yeast tRNA as a negativecontrol.

neu3 is widely expressed, with highest levels in immune tissues spleenand thymus, and in the lung (FIG. 14). Total RNA was isolated from theindicated mouse brain regions and non-neural tissues and the levels ofneu3 transcripts were analyzed by RNase protection assays. cRNA probesthat were used for detection of neu3 transcripts covered the regionencoding the NHR of mouse neu3. Specific protected fragments areindicated on the left of the panel. E, Embryonic day; P, postnatal day;AD, adult; HC, hippocampus; CTX, cerebral cortex; OLF, olfactory bulb;STR, striatum; THA, thalamus; COL, colliculi; MID, midbrain; CBL,cerebellum; PONS, pons; MED, medulla; LIV, liver; HEA, heart; KID,kidney; LUN, lung; MUS, muscle; SPL, spleen; TES, testis; THY, thymus;tRNA, yeast tRNA as a negative control.

Expression of neu4 was detected only in muscle and heart.

EXAMPLE 7 Isolation and Characterization of Factors Interacting withNeu1

An adult rat brain library was screened using a yeast two hybrid systemwith a mouse Neu1 protein as a ligand with which to isolate and identifyproteins that interact with Neu1. Fifty-three (53) clones were isolated.cDNAs that yielded more than 1 clone were sequenced and identified asfour interactors: NeuI-1, NeuI-2, NeuI-3, and NeuI-4.

Sequence analyses showed that all interactors were novel proteins andcontain RING finger domain located in the C-terminus of the protein.NeuI-1 (4 clones) is a novel splice variant (SEQ. ID. NO.: 29) of zincfinger protein Miz1/PIASX/ARIP3 (GenBank accession number NM_(—)008602;AF077953; AF077954; AF044058). NeuI-2 (3 clones) is a fourth homologue(SEQ. ID. NO.: 30; GenBank accession number AF277171; AF302084) of zincfinger protein ZNF127 (GenBank accession number U19106; U19107), NeuI-3(9 clones) has highest homology to a human hypothetical protein (GenBankaccession number AK001459) and to a Drosophila hypothetical protein(AAF56052.2) produced from CG4813 gene of a genomic scaffold (GenBankaccession number AE003740) (SEQ. ID. NO.: 31), and NeuI-4 (12 clones) isthe homologue of the androgen receptor co-activator ARA54 (SEQ. ID. NO.:32; GenBank accession number AF060544).

Expression of neuI-1-neuI-4 mRNAs was analyzed in developing and adultrat brain and non-neural tissues (FIG. 15). Total RNA was isolated fromthe indicated rat tissues and the levels of transcripts were analyzed byRNase protection assays with cRNA probes specific for NeuI-1, NeuI-2,NeuI-3 and NeuI-4 transcripts. Specific protected fragments areindicated on the left of each panel. Bottom panel shows the levels ofGAPDH mRNA in the RNA samples. E, Embryonic day; P, postnatal day; ad,adult; cbl, cerebellum; ctx, cerebral cortex; stem, brainstem; hc,hippocampus; hc+KA, hippocampus from rats treated for 4h with theglutamate receptor agonist kainic acid; hea, heart; kid, kidney; mus,skeletal muscle; spl, spleen; thy, thymus; tes, testis; li, liver; lu,lung; tRNA, yeast tRNA as a negative control.

All the identified Neu1 interactors were expressed in the brain andskeletal muscle, tissues where neu1 mRNA is predominantly expressed.This suggests that neuI-1-neuI-4 interact with Neu 1 protein and modifyits activity in vivo.

EXAMPLE 8 Neu1 Affects the Activation of Immediate Early Genes (IEGs)

The function of Neu1 as a calcium-dependent transcriptional repressorwas studied by transfecting Neuro2A cells with m-neu1-CMV expressionvector. This system was used to examiner whether or not Neu1 couldsuppress transcription of IEGs in response to the simultaneousstimulation of cells with calcium, ionophore A23187, and forskolin. As acombination of forskolin with calcium, ionophores leads to increasedc-fos mRNA levels in Swiss 3T3 cells (Mehmet H, 1990). Because theinduction of immediate early genes by Ca²⁺ influx requirescAMP-dependent protein kinase in PC12 cells (Ginty DD, 1991), it wasdecided to apply the forskolin and ionophore co-treatment to Neuro-2Acells.

Upon transient transfection of m-Neu1 into Neuro2A cells, activation oftranscription of several IEGs (c-fos, junB, junD, c-jun, fra-1, andfra-2) from their endogenous promoters was significantly reduced inresponse to the raised intracellular Ca²⁺ (Ca ionophore) and cAMPactivity (forskolin) levels. Neu1 elevated levels affected the amplitudeof the IEG mRNA induction, however, had no effect on the time course ofinduction. This finding couples Neu1 to stimulus (calcium)-dependenttranscriptional regulation.

EXAMPLE 9 Interaction with TBP

Because m-Neu1 affects a variety of target gene promoters in transienttransfection assays, it was hypothesized that m-Neu1 functions byinterfering directly with the function of Pol II complex, particularlysuppressing the TBP transcriptional activity. To examine thispossibility, m-Neu-CMV expression constructs were cotransfected togetherwith m-TBP-CMV. The effect of this procedure on thymidine kinase (tk)promoter was examined. Increasing amounts of Neu-CMV while mTBP-CMVamounts were kept constant (and vice versa) resulted in the decreasedactivity of the reporter gene in Neuro2A cells. These results suggestedthat m-Neu1 could repress transcription by direct interaction with TBP.

EXAMPLE 10 Neoplastic Diagnostic Assay Using Genomic DNA

A biopsy is obtained from a subject possibly suffering from a neoplasticdisease, such as an astrocytoma. Following excision of the sample, thetissue is immediately minced and quickly frozen in liquid nitrogen. Asample 1 gram sample of tissue is then ground with a prechilled mortarand pestle for suspension. The ground tissue is then suspended inapproximately 1.2 ml of digestions buffer (100 mM NaCl, 10 mM Tris-Cl,pH 8.0, 25 mM EDTA, pH 8.0, 0.5% (w/v) SDS, and 0.1 mg/ml proteinase K,which is added fresh for each use). Samples are shaken and incubated for12 to 18 hours at 50° C.

Following the incubation period, the samples are extracted with an equalvolume of phenol/chloroform/isoamyl alcohol, to remove proteinatiousmaterial. Centrifuge the mixture for 10 minutes at 1700×g. If phases donot resolve well, add another volume of digestion buffer, omittingproteinase K, and repeat centrifugation. Repeat the extraction until nothick white material appears at the interface. Transfer the top aqueouslayer to a new tube.

To this tube is added ½ volume of 7.5 M ammonium acetate and 2 volumesof 100% ethanol. This mixture is centrifuged for 2 minutes at 1700×g.Following the centrifugation, the resulting pellet is washed with 70%ethanol, and then air dried and will be resuspended in Tris-EDTA bufferat approximately 1 mg/ml.

To prevent shearing of high molecular weight DNA it may be advisable toremove organic solvents and salt by two dialyses against 100 volumes ofTris-EDTA buffer for more than 24 hours. If this step is performed, thepellet is not resuspended in Tris-EDTA buffer.

The purified DNA is analyzed for the presence or mutation of a wild typecopy of a neu family gene using Southern Blotting and sequencing. Theabsence or mutation of such in the subject indicates the presence of amalignancy.

EXAMPLE 11 Neoplastic Diagnostic Assay Using RNA Analysis

A tissue sample is obtained from a subject possibly suffering from aneoplastic disease. The biopsy is removed and cut into less than 2 grampieces. These pieces are quick-frozen in liquid nitrogen. Twenty (20 ml)of tissue guanidinium solution is used to process 2 grams of tissue. Thetissue guanidinium solution is prepared by dissolving 590.8 grams ofguanidinium isothiocyanate in approximately 400 ml DEPC-treated H₂O. Tothis is added 25 ml of 2M Tris-Cl, pH 7.5 (0.05 M final) and 20 ml of0.5 M Na₂EDTA, pH 8.0 (0.01 M final). Stir overnight, adjust the volumeto 950 ml, and filter. Finally, add 50 ml 2-ME.

Once the tissue guanidinium solution is added to the tissue, the sampleis immediately ground in a tissuemizer with two or three ten secondbursts. Following disruption, the solution is subjected tocentrifugation for 10 minutes at 12,000×g in a SS-34 rotor, at 12° C. Tothe supernatant is added 0.1 volumes of 20% Sarkosyl. This mixture isthen subjected to heat at 65° C., for 2 minutes.

To this heated solution is added 0.1 grams of CsCl/ml of solution, whichis mixed until it dissolves. The sample is next layered over 9 ml of5.7M CsCl in silanized and autoclaved SW-28 tubes. These tubes arecentrifuged overnight at 113,000×g in a SW-28 rotor at 22° C.

Following the centrifugation step, the supernatant is removed and thetubes are inverted to drain. The bottom of each tube is removed and theRNA pelleted contained therein is placed in a 50-ml plastic tube. Three(3) ml of tissue resuspension buffer and allow pellet to resuspendovernight or longer at 4° C. Extract solution sequestially with 25:24:1phenol/chloroform/isoamyl alcohol, then with 24:1 chloroform/isoamylalcohol. Add 0.1 volume of 3 M sodium acetate, pH 5.2, and 2.5 volume of100% ethanol, precipitate, and resuspend RNA in water. The sample isquantitated and analyzed for the expression of the neu family of genes.Samples with aberent expression levels indicate the presence ofneoplastic cells.

EXAMPLE 12 Ex Vivo Exogenous Gene Expression

Cells are isolated from a subject to be transfected with a constructencoding Neu protein (Neu construct). The Neu construct is transfectedusing DEAE-dextran. The cells are seeded in 6-well tissue culture platesand are transfected with a total of 1-2 μg of total DNA containing theNeu contruct. After 5 hours, 1 ml of DMEM containing 20% fetal bovineserum is added and the cells are allowed to incubate overnight.

Expression of Neu is then determined using immunoprecipitation with anantibody-agarose conjugate (8 μg) plus 25 μl of a 50% slurry of ProteinA-agarose. Immune complexes are washed in a wash buffer (20 mM Hepes, pH7.4, 10 mM MgCl₂, and 1 mM DTT). The immune complexes are placed in adenaturing protein sample buffer that separated the antibody from anybound antigen. The protein samples are then run on SDS-polyacrylamidegel electrophoresis to detect the expression of a target Neu protein.

The foregoing description details certain embodiments of the invention.It will be appreciated, however, that no matter how detailed theforegoing appears in the text, the invention can be practised in manyways. Although this invention has been described in terms of certainpreferred embodiments, other embodiments which will be apparent to thoseof ordinary skill in the art in view of the disclosure herein are alsowithin the scope of this invention. As is also stated above, it shouldbe noted that the use of particular terminology when describing certainfeatures or aspects of the invention should not be taken to imply thatthe terminology is being re-defined herein to be restricted to includeany specific characteristics of the features or aspects of the inventionwith which that terminology is associated. Accordingly, the scope of theinvention should therefore be construed in accordance with the appendedclaims and any equivalents thereof.

1-30. (canceled)
 31. A purified neuralized (Neu) polypeptide, comprisingat least one neuralized homology repeat domain and a C3HC4 RING-zincfinger domain, wherein said Neu polypeptide has at least 90% sequenceidentity to SEQ ID NO:22 and is capable of suppressing transcription.32. The purified Neu polypeptide according to claim 31, wherein said Neupolypeptide has at least 95% sequence identify to SEQ ID NO:22.
 33. Thepurified Neu polypeptide according to claim 31, wherein said Neupolypeptide comprises SEQ ID NO:22.
 34. The purified Neu polypeptideaccording to claim 31, wherein said neuralized homology repeat domaincomprises SEQ ID NO:48.
 35. An antibody capable of specifically bindingto an isolated Neu polypeptide according to claim
 31. 36. The antibodyaccording to claim 35, wherein said antibody specifically binds to apolypeptide comprising at least 10 consecutive amino acids of said Neupolypeptide.
 37. The antibody according to claim 35, wherein saidantibody is a monoclonal antibody.